Liying Huang

Screening and quantitative analysis of antioxidants in the fruits of Livistona chinensis R. Br using HPLC-DAD–ESI/MS coupled with pre-column DPPH assay

In this study, a high performance liquid chromatography-photo diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI-MS) with pre-column DPPH assay is developed for screening the antioxidant components in the fruits of Livistona chinensis R. Br. Accordingly, six antioxidative flavonoids are identified as orientin, isoorientin, vitexin, isovitexin, isorhamnetin-3-O-glucoside and tricin in methanolic extract of L. chinensis fruits, based on their mass spectra and fragmentation patterns. To the best of our knowledge, orientin, isoorientin, isovitexin and isorhamnetin-3-O-glucoside were found firstly in this plant. The free radical scavenging activity of the six antioxidants found is further examined by off-line DPPH assay. The results indicated that the free radical scavenging activity of orientin and isoorientin are stronger than those of two antioxidative drugs, vitamin C and baicalin. In addition, an HPLC-DAD method is firstly established for simultaneous determination of the six antioxidants in L. chinensis fruits. Tricin was found to be the major component in L. chinensis fruits.

Chromatographic fingerprint and quantitative analysis of seven bioactive compounds of Scutellaria barbata.

Scutellaria barbata D. Don is widely used as a folk antitumor and anti-inflammatory agent in Asia. However, a simple and global quality control method for S. barbata was lacking. In this study, six phenolic compounds, including P-coumaric acid, scutellarin, apigenin 5-O-β-glucopyranoside, luteolin, apigenin and 4-hydroxywogonin were obtained from S. barbata by phytochemical investigations. The six compounds plus baicalein show cytotoxcities to the nine human cancer cells, K562, MGC-803, HL60, SH-SY5Y, SW1116, SMMC-7221, SW480, HepG2 and KB. Subsequently, a high-performance liquid chromatography with photodiode array detector (HPLC-DAD) was developed for both fingerprint analysis of S. barbata and quantitative determination of the seven anticancer active compounds in S. barbata. The chromatographic separation was accomplished on an Ultimate™ XB-C18 column (4.6 x 250 mm, 5 µm) in 65 min. For fingerprinting, 26 common peaks were found and selected as characteristic peaks to assess the consistency of S. barbata samples. For quantitative analysis, the seven bioactive compounds showed good regression relationship ( R² > 0.999) within test ranges and the recovery of the method was in the range of 90-105 %. In brief, the present study provides the fingerprint analysis and quantitative methods for global and systematical quality control of S. barbata for its anticancer usage.

Preparative isolation of six anti-tumour biflavonoids from Selaginella Doederleinii Hieron by high-speed counter-current chromatography

INTRODUCTIONnBiflavonoids are the primary constituents of Selaginella doederleinii Hieron, to which different bioactivities have been attributed, including anti-cancer, anti-inflammatory, anti-oxidant, anti-fungal and anti-virus activity. However, effective methods for separation of these compounds are not currently available.nnnOBJECTIVEnTo develop a high performance and bioassay-guided method for preparative isolation of biflavonoids from S. doederleini via high-speed counter-current chromatography (HSCCC).nnnMETHODSnThe anti-proliferation effects of four fractions (70% ethanol, petroleum ether, dichloromethane and acetic ether extracts) of S. doederleinii on five human cancer cells were monitored. The dichloromethane and acetic ether extracts showed good cytotoxicities to the studied cancer cell lines, guiding the subsequent separation. Two solvent systems composed of n-hexane:ethyl acetate:methanol:water (1:2:1.5:1.5, v/v) and n-hexane:ethyl acetate:methanol:water (3:2:3:2, v/v) were developed for separation of the active fractions, respectively. Identification of the biflavonoids was performed by EI-MS(n) , (1) H- and (13) C-NMR.nnnRESULTSnUnder the optimised conditions, 12.6 mg amentoflavone (91.4%), 6.6 mg robustaflavone (90.4%), 7.5 mg 2, 3-dihydro-3, 3-biapigenin (98.2%) and 7.3 mg 3, 3-binaringenin (90.3%) from acetic ether extract (500 mg) and 6.3 mg heveaflavone (93.5%) and 5.3 mg 7, 4, 7, 4-tetra-O-methyl-amentoflavone (94.5%) from dichloromethane extract (200 mg) were obtained, respectively. The anti-proliferation effects of the six biflavonoids on the five human cancer cells were further verified.nnnCONCLUSIONnThe study provides methodological references for simultaneously preparative isolation of several bioactive biflavones from the herbal family of Selaginella. It is the first report discovering 2, 3-dihydro-3, 3-biapigenin and 3, 3-binaringenin from this herb and describing their cytotoxicities to human cancer cell lines.

Promotion proliferation effect of a polysaccharide from Aloe barbadensis Miller on human fibroblasts in vitro.

A polysaccharide fraction was isolated from fresh Aloe barbadensis Miller leaves, which can promote the wound healing of the superficial II scald model mice. The monosaccharide composition and linkage determination were investigated by methylation and GC-MS, acetylation and GC, 13C NMR and DEPT. The results show that its glycosyl components contain D-glucose, D-galactose, D-xylose in a molar ratio of 5:5:1, and the API consists of a backbone of -->2)-alpha-D-Galp-(1-->2)-alpha-D-Glcp-(1-->, having a branch of alpha-D-xylofuranosyl residue that is (1-->3) linkage at O-3 of alpha-D-galactopyranosyl residue. It was found that the API could enhance proliferation of the human fibroblasts in vitro. The mechanisms of promotion proliferation were studied preliminarily.

Study of the electrochemical behavior of isorhamnetin on a glassy carbon electrode and its application

The electrochemical behavior of isorhamnetin (ISO) at a glassy carbon electrode was studied in a phosphate buffer solution (PBS) of pH 4.0 by cyclic voltammetry (CV) and differential pulse voltammetric method (DPV). A well-defined redox wave of ISO involving one electrons and one proton appeared. The electrode reaction is a reactant weak adsorption-controlled process with a charge transfer coefficient (alpha) of 0.586. Based on the understanding of the electrochemical process of ISO at the glassy carbon electrode, analysis of ISO can be realized. Under optimal conditions, the oxidation peak current showed linear dependence on the concentration of ISO in the range of 1.0x10(-8) to 4.0x10(-7)M and 1.0x10(-6) to 1.0x10(-5)M. The detection limit is 5.0x10(-9)M. This method has been successfully applied to the detection of ISO in tablets.

Ethyl acetate extract from Selaginella doederleinii Hieron inhibits the growth of human lung cancer cells A549 via caspase-dependent apoptosis pathway

ETHNOPHARMACOLOGICAL RELEVANCEnSelaginella doederleinii Hieron has been used as a folk medicine for the treatment of different cancers, especially for nasopharyngeal carcinoma, lung cancer and trophoblastic tumor in China. Previously, the ethyl acetate extract from S. doederleinii (SDEA extract) showed favorable anti-cancer potentials. However, the main chemical composition and anticancer mechanism of the SDEA extract were still not very clear. Until now, there are no reports available about the oral toxicity of the extract.nnnAIM OF STUDYnThe present study was to further elucidate the chemical composition and anti-lung cancer mechanism of the SDEA extract, and evaluate the acute oral toxicity of the extract.nnnMATERIALS AND METHODSnThe SDEA extract was separated and analysed by HPLC to disclose its main chemicals. The effects of the extract were then investigated in vitro on cell viability, apoptosis and cell cycle using fluorescence microscopy and flow cytometry, and the molecular mechanism against human lung cancer cells A549 was further studied by western blot assays. The in vivo anti-cancer effect of the extract was evaluated in A549 xenograft mice model by histochemical assay, and tumor growth, microvascular density (MVD) and Ki67 expression were also measured. In addition, acute oral toxicity test of the extract was executed in mice.nnnRESULTSnSDEA extract mainly contained eight biflavonoids. The extract caused the loss of mitochondrial membrane potential and induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating caspase-9 and caspase-3 and blocked the cell cycle in S phase. The extract reduced expression of antigen Ki67, decreased MVD, and significantly inhibited the tumor growth. The extract did not show apparent oral acute toxicity in healthy mice.nnnCONCLUSIONnThe SDEA extract exerted anti-tumor effect through activating mitochondrial pathways and reducing Ki67 expression and MVD. Low oral acute toxicity suggested it a promising chemotherapy agent.

Determination of Seven Biflavones of Selaginella Doederleinii by High Performance Liquid Chromatography

Selaginella doederleinii Hieron (S. doederleinii) is widely used as an antitumor and anti-inflammatory agent in Asia, and is famous for containing biflavonoids, which have been reported as the bio-active constituents possessing anticancer and anti-oxidant effects. However, a simple and systematic quality control method for determining biflavonoids in S. doederleinii is lacking. In this study, seven biflavones, including amentoflavone, robustaflavone, 2″, 3″-dihydro-3′, 3″′-biapigenin; 3′, 3″′-binaringenin, delicaflavone, heveaflavone, and 7, 4′, 7″, 4″′-tetra-O-methyl-amentoflavone, were obtained from S. doederleinii. These seven compounds that are present in high concentration were used as detection markers for development of a quality control method by HPLC with a UV-visible detector. The chromatographic separation was accomplished on a C18 column with a mobile phase composed of acetonitrile–water (0.5% acetic acid) in 52 min. With the optimized conditions, the seven biflavones showed good regression relationships (R2 > 0.9989) within the test range and the recovery was in the range of 90.1–102.5%. The LODs of the seven analytes were between 0.2 and 1.17 µg/mL. The method was also validated for precision and accuracy and was successfully applied to the determination of these compounds in six batches of S. doederleinii from different areas. In conclusion, the present study provides a systematic and useful method for quality evaluation of S. doederleinii.

Electrocatalytic oxidation and determination of norepinephrine in the presence of ascorbic and uric acids at a poly (Evans Blue)—modified glassy carbon electrode

A sensitive and selective electrochemical method for the determination of norepinephrine using a poly (Evans Blue) film-modified glassy carbon electrode was developed. The polymer film-modified electrode shows excellent electrocatalytic activity toward the oxidation of norepinephrine (NE) in phosphate buffer solution (pH 5.0). The linear range of 5.0 × 10−7–1.8 × 10−5 M and detection limit of 3.5 × 10−8 M were observed for the determination of NE in pH 5.0 phosphate buffer solutions. The interference studies showed that the modified electrode had excellent selectivity for the determination of NE in the presence of large excess of ascorbic acid (AA) and uric acid (UA). The differences of the oxidation peak potentials for NE-AA and NE-UA were about 175 and 172 mV, respectively. The resolution is large enough to determine AA, NE and UA individually. This work provides a simple and easy approach to selective detection of NE in the presence of AA and UA in physiological samples.

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.Key messagesDelicaflavone showed anti-lung cancer effects in vitro and in vivo.Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway.Delicaflavone did not show observable side effects in a xenograft mouse model.Delicaflavone may represent a potential therapeutic agent for lung cancer.

A strategy for integrated pharmacokinetic study of cardiovascular herbal medicines based on chemiluminescence and HPLC-MS/MS assays: a case using Danshen injection

Currently, it is important to explore any feasible tool to represent the integrated in vivo process of a traditional Chinese medicine (TCM) to understand its clinical dosage regimen from ancient empirical therapy. Herein, we put forward a strategy to characterize the integrated pharmacokinetic properties of multiple-component cardiovascular herbal medicines with a flow injection chemiluminescence (FI-CL) technique and an HPLC-MS/MS assay. Taking Danshen injection as a carrier, the main ingredients of this preparation were determined by HPLC as danshensu sodium (DSSN, 554.13xa0mg g−1), protocatechuic aldehyde (PCAL, 40.55 mg g−1), rosmarinic acid (RA, 21.66 mg g−1), salvianolic acid B (SAB, 1.67 mg g−1) and salvianolic acid A (SAA, 68.60 mg g−1). This was followed by confirmation that these phenolics could inhibit the CL signal of the KIO4–luminol system and that the inhibition was closely correlative with both their concentrations and antioxidative capability. Further, the protective effects of the main components (40 mg kg−1 per day, i.v. for 7 days) on isoprenaline-induced myocardial injury in mice were evaluated, confirming that these phenolics were the main effective substances and that antioxidation was the most important of their therapeutic effects. Subsequently, the PK profiles of the main phenolic acids in rat plasma (21 mg kg−1, single i.v.) were studied by HPLC-MS/MS with a multiple reaction monitoring mode. The phenolic acid mixture solutions could be prepared in distilled water according to their plasma drug concentrations to simulate the plasma sample solutions at each studied PK time point. All these provided the basis to evaluate their integrated PK properties through determining the total phenolic acid content in the simulation solutions by the inhibited CL signal with salvianolic acid A (SAA), the most powerful ingredient in Danshen injection, as a reference. Finally, the integrated PK properties can be characterized into the SAA “equivalent” parameters, including t1/2β, k, and AUC(0–∞), by the inhibited CL signal. Also, the results were compared with those from the “plasma drug concentration sum method” and the “AUC weighting integrated method” from previous reports. In summary, the present study provides a feasible strategy for PK analysis of cardiovascular herbal medicines with antioxidants as the main effective substances.