Activation of mitochondrial-associated apoptosis signaling pathway and inhibition of PI3K/Akt/mTOR signaling pathway by voacamine suppress breast cancer progression.

BACKGROUND: Breast cancer is one of the malignant tumors with the highest morbidity and mortality rate. Numerous efficient anti-breast cancer drugs are being derived from the development of natural products. Voacamine (VOA), a bisindole alkaloid isolated from Voacanga africana Stapf, possesses various pharmacological and biological activities. PURPOSE: In this study, we investigated the efficacy of VOA against breast cancer cells and elucidated the underlying mechanisms in vitro and in vivo. METHODS: Human breast cancer cell line MCF-7 and mouse breast cancer cell line 4T1 were used to study the underlying anti-cancer mechanisms of VOA. The proliferation was detected by MTT, colony formation, cell proliferation and wound-healing migration assays. Flow cytometry was utilized to determine the level of reactive oxygen species (ROS) cell-cycle, apoptosis and mitochondrial membrane potential. The target proteins were analyzed by Western blot. Molecular docking was performed and scored by AutoDock. Subcutaneous cancer models in mice were established to evaluate the anticancer effects in vivo. RESULT: Our results demonstrated that VOA selectively suppressed breast cancer MCF-7 and 4T1 cells proliferation with IC50 values of 0.99 and 1.48 µM, and significantly inhibited the migration and colony formation of tumor cells. Furthermore, the cell cycle was arrested in the S phase with the decreased expression levels of CDK2, Cyclin A and Cyclin E. Additionally, exposure to VOA dose-dependently brought about dose-dependently the loss of mitochondrial membrane potential (Δψm) and amassment of reactive oxygen species (ROS), resulting in the initiation of the intrinsic apoptotic pathway. Western blot analysis unveiled that VOA significantly activated mitochondrial-associated apoptosis and obviously suppress the PI3K/Akt/mTOR pathway via modulation of related protein expression levels in both tumor cell lines. In tumor-bearing mouse models, administration of VOA dose-dependently inhibited the tumor growth without causing apparent toxicities. CONCLUSION: These findings revealed the novel properties of VOA in promoting apoptosis of breast cancer cells by activating mitochondrial-associated apoptosis signaling pathway and inhibiting PI3K/Akt/mTOR signaling pathway and significantly decreasing tumor size without detecting appreciable toxicity. In summary, the present results demonstrated VOA could be an encouraging drug candidate to cure breast cancer, exhibiting an effective method to exploit unique drugs from natural components.

Fish Oil Is More Potent than Flaxseed Oil in Modulating Gut Microbiota and Reducing Trimethylamine-N-oxide-Exacerbated Atherogenesis.

Trimethylamine-N-oxide (TMAO) is a risk factor for atherosclerosis. We compared the potency of fish oil with flaxseed oil in reducing TMAO-exacerbated atherogenesis. Five groups of ApoE-/- mice were given one of five diets, namely, a low-fat diet, a Western high fat diet (WD), a WD plus 0.2% TMAO, and two WDs containing 0.2% TMAO with 50% lard being replaced by flaxseed oil or fish oil. TMAO accelerated atherosclerosis and disturbed cholesterol homeostasis. Compared with flaxseed oil, fish oil was more effective in inhibiting TMAO-induced atherogenesis by lowering plasma cholesterol and inflammatory cytokines. Both oils could reverse TMAO-induced decrease in fecal acidic sterols. Fish oil promoted fecal output of neutral sterols and downregulated hepatic cholesterol biosynthesis. Fish oil was more effective than flaxseed oil in promoting the growth of short-chain fatty acid-producing bacteria and lowering microbial generation of lipopolysaccharide. In conclusion, fish oil is more potent than flaxseed oil to ameliorate TMAO-exacerbated atherogenesis.

Comprehensive Quantitative Analysis of 32 Chemical Ingredients of a Chinese Patented Drug Sanhuang Tablet.

Sanhuang Tablet (SHT) is a Chinese patented drug commonly used for the treatment of inflammations of the respiratory tract, gastrointestinal tract, and skin. It contains a special medicinal composition including the single compound berberine hydrochloride, extracts of Scutellariae Radix and Rhei Radix et Rhizoma, as well as the powder of Rhei Radix et Rhizoma. Despite advances in analytical techniques, quantitative evaluation of a Chinese patented drug like SHT remains a challenge due to the complexity of its chemical profile. In this study, ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to simultaneously quantify 29 non-sugar small molecule components of SHT (11 flavonoids, two isoflavonoids, one flavanone, five anthraquinones, two dianthranones, five alkaloids, two organic acids and one stilbene). Three major saccharide components, namely fructose, glucose, and sucrose, were also quantitatively determined using high performance liquid chromatography-charged aerosol detector (HPLC-CAD) on an Asahipak NH2P-50 4E amino column. The established methods were validated in terms of linearity, sensitivity, precision, accuracy, and stability, and then successfully applied to analyze 27 batches of commercial SHT products. A total of up to 57.61% (w/w) of SHT could be quantified, in which the contents of the determined non-saccharide small molecules varied from 5.91% to 16.83% (w/w) and three saccharides accounted for 4.41% to 48.05% (w/w). The results showed that the quality of the commercial products was inconsistent, and only four of those met Chinese Pharmacopoeia criteria.

Comprehensive quantitative analysis of Chinese patent drug YinHuang drop pill by ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry.

YinHuang drop pill (YHDP) is a new preparation, derived from the traditional YinHuang (YH) decoction. Since drop pills are one of the newly developed forms of Chinese patent drugs, not much research has been done regarding the quality and efficacy. This study aims to establish a comprehensive quantitative analysis of the chemical profile of YHDP. ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify 34 non-sugar small molecules including 15 flavonoids, 9 phenolic acids, 5 saponins, 1 iridoid, and 4 iridoid glycosides in YHDP samples, and 26 of them were quantitatively determined. Sugar composition of YHDP in terms of fructose, glucose and sucrose was examined via a high performance liquid chromatography-evaporative light scattering detector on an amide column (HPLC-NH2P-ELSD). Macromolecules were examined by high performance gel permeation chromatography coupled with ELSD (HPGPC-ELSD). The content of the drop pill's skeleton component PEG-4000 was also quantified via ultra-high performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD). The results showed that up to 73% (w/w) of YHDP could be quantitatively determined. Small molecules accounted for approximately 5%, PEG-4000 represented 68%, while no sugars or macromolecules were found. Furthermore, YHDP showed no significant differences in terms of daily dosage, compared to YinHuang granules and YinHuang oral liquid; however, it has a higher small molecules content compared to YinHuang lozenge.

[Chemical variation in Aurantii Fructus before and after processing based on UHPLC-Q-TOF-MS].

To explore the processing mechanism of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong by analysing the chemical variation between raw and processed Aurantii Fructus with different methods based on UHPLC-Q-TOF-MS. The total ion chromatograms detected in positive and negative ion modes, and ion peak area ratio before and after processing were taken as variation indexes in the comparison. The results indicated that fermented Aurantii Fructus could produce three new ingredients, namely eriodictyol-7-glucoside, hesperetin-7-O-glucoside and 5-demethylnobiletin. At the same time, it could significantly increase the content of naringenin and hesperetin components, and could increase the content of such limonin derivatives as sudachinoid A, obacunoic acid and limoninand nomilinic acid. This suggests that the fermentation processing method of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong is of important significance for enhancing biological activity and bioavailability, and improving the clinical efficacy of Aurantii Fructus decoction pieces, and so is worth further protection and promotion.

Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of Ma-Zi-Ren-Wan.

Ma-Zi-Ren-Wan (MZRW) is a classic Chinese formula which has been used to treat human constipation in China for over 2000 years. In order to make good and rational use of this formula in the future, this paper presents the first attempt to track the pharmacokinetic features of MZRW in rat using rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Ten chemical components of MZRW, namely, rhein, emodin, aloe emodin, hesperidin, naringin, amygdalin, albiflorin, paeoniflorin, magnolol and honokiol, were simultaneously determined in rat plasma after a single oral administration (10g/kg body weight) of MZRW to rats. Geniposide and liquiritin were used as internal standards. The separation was performed on a Waters ACQUITY BEH C18 column (100mm×2.1mm, 1.7µm). The detection was conducted by multiple-reaction monitoring (MRM) in negative ionization mode. Two highest abundant MRM transitions without interference were optimized for each analyte. This method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r(2)>0.995) over the concentration range from 3.9 to 125.0ng/mL for emodin, 3.9-500.0ng/mL for amygdalin, 2.0-4000.0ng/mL for naringin and hesperidin, 3.9-2000.0ng/mL for magnolol, 7.8-2000.0ng/mL for rhein and 3.9-4000.0ng/mL for albiflorin, paeoniflorin, aloe emodin and honokiol. The intra-day and inter-day precision (relative standard deviation) was within 15%, the accuracy (relative error) ranged from -13.6% to 15.1%, and the lower limit of quantification in plasma ranged between 2.0ng/mL and 7.8ng/mL. Extraction recovery, matrix effect and stability were satisfactory. The validated method was successfully applied to a pharmacokinetic study of these ten compounds after oral administration of MZRW to rats. The pharmacokinetic parameters of each compound can facilitate clinical studies in the future.

Comprehensive quantitative analysis of Shuang-Huang-Lian oral liquid using UHPLC-Q-TOF-MS and HPLC-ELSD.

Shuang-Huang-Lian oral liquid (SHL) is a well-known Chinese patent drug containing three herbal medicines: Radix Scutellariae, Flos Lonicerae Japonicae and Fructus Forsythiae. It is usually used to treat acute upper respiratory tract infection caused by virus or bacteria. Although the licensing of botanical drug Veregen approved by FDA has indicated the importance of quantitative analysis in quality control of herbal medicines, quantitative evaluation of a Chinese patent drug like SHL remains a challenge due to the complex chemical profile. In this study, 15 small molecular components of SHL (four flavonoids, six quinic acid derivatives, three saponins and two phenylethanoid glycosides) were simultaneously determined using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The contents of the three major saccharides, namely fructose, glucose and sucrose were quantified using high performance liquid chromatography-evaporative light scattering detector on an amino column (HPLC-ELSD). The macromolecules were quantified by precipitating in 80% ethanol, drying the precipitate, and then weighing. The established methods were validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to analyze 12 batches of commercial products of SHL produced by four different manufacturers. The results indicated that 57.52-78.11% (w/w) of SHL could be quantitatively determined (non-saccharide small molecules: 1.77-3.75%, monosaccharides: 0.93-20.93%, macromolecules: 2.63-5.76% and sucrose: 49.20-65.94%). This study may provide a useful way to comprehensively evaluate the quality of SHL.

Histochemical evaluation of alkaloids in rhizome of Coptis chinensis using laser microdissection and liquid chromatography/mass spectrometry.

Traditional macroscopic and microscopic identification methods of medicinal materials are economical and practical, but usually experience-based due to few chemical supports. Here histochemical evaluation on bioactive components of Coptidis Rhizoma (CR) in anatomic sections using laser microdissection and liquid chromatography-mass spectrometry (LMD-LC-MS) was developed to correlate the inner quality and outer features of materials from different growing areas. Results of a total 33 peaks representing potential different alkaloids were detected and 8 common peaks were identified as the major alkaloids, namely magnoflorine, thalifendine, columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine. Six major alkaloids were quantified in the top and middle sections of raw materials and in their tissues and cells at the same time. Histochemical analyses showed consistent results with direct determination in raw materials and explained the reason why top sections of all samples contained higher contents of alkaloids by giving out attributions of each alkaloid in different anatomic sections. Besides, results manifested the distribution and accumulation rules of alkaloids in diverse tissues and cells of CR. This study demonstrates an effective and scientific way to correlate bioactive components and morphological features of medicinal materials, which is beneficial to future research, agriculture and application.

Unconjugated bilirubin mediates heme oxygenase-1-induced vascular benefits in diabetic mice.

Heme oxygenase-1 (HO-1) exerts vasoprotective effects. Such benefit in diabetic vasculopathy, however, remains unclear. We hypothesize that bilirubin mediates HO-1-induced vascular benefits in diabetes. Diabetic db/db mice were treated with hemin (HO-1 inducer) for 2 weeks, and aortas were isolated for functional and molecular assays. Nitric oxide (NO) production was measured in cultured endothelial cells. Hemin treatment augmented endothelium-dependent relaxations (EDRs) and elevated Akt and endothelial NO synthase (eNOS) phosphorylation in db/db mouse aortas, which were reversed by the HO-1 inhibitor SnMP or HO-1 silencing virus. Hemin treatment increased serum bilirubin, and ex vivo bilirubin treatment improved relaxations in diabetic mouse aortas, which was reversed by the Akt inhibitor. Biliverdin reductase silencing virus attenuated the effect of hemin. Chronic bilirubin treatment improved EDRs in db/db mouse aortas. Hemin and bilirubin reversed high glucose-induced reductions in Akt and eNOS phosphorylation and NO production. The effect of hemin but not bilirubin was inhibited by biliverdin reductase silencing virus. Furthermore, bilirubin augmented EDRs in renal arteries from diabetic patients. In summary, HO-1-induced restoration of endothelial function in diabetic mice is most likely mediated by bilirubin, which preserves NO bioavailability through the Akt/eNOS/NO cascade, suggesting bilirubin as a potential therapeutic target for clinical intervention of diabetic vasculopathy.

A novel and rapid HPGPC-based strategy for quality control of saccharide-dominant herbal materials: Dendrobium officinale, a case study.

Qualitative and quantitative characterization of natural saccharides, especially polysaccharides, in herb materials remains a challenge due to their complicated structures and high macromolecular masses. Currently available methods involve time-consuming and complicated operations, and present poor specificity. Here, a novel and rapid high-performance gel permeation chromatography (HPGPC)-based approach is described for quality assessment of saccharide-dominant herbal materials by simultaneous qualitative and quantitative analysis of saccharide components. Dendrobium officinale, one of the rarest tonic herbs worldwide, was employed as the model herb in this study. First, a HPGPC fingerprint based on the molecular weight distribution of its carbohydrate components was established for qualitative identification of D. officinale. Then, HPGPC-guided dominant holistic polysaccharide marker was separated using ultra-filtration followed by HPGPC determination for quantitative evaluation of D. officinale. The experimental results suggest that this method is more efficient, stable, and convenient compared with the currently available methods for authentication and quality evaluation of D. officinale, and we expect the method will have similar advantages when used for quality control of other saccharide-dominant herbal materials and products.

Cyclooxygenase-2-dependent oxidative stress mediates palmitate-induced impairment of endothelium-dependent relaxations in mouse arteries.

Palmitic acid, one of the saturated free fatty acids, impairs cardiovascular function as manifested by inducing vascular inflammation, apoptosis and over-production of reactive oxygen species (ROS) although the origin for ROS remains unclear. The present study investigated the cellular mechanisms underlying palmitate-induced impairment of endothelial function. Ex vivo treatment in tissue culture with palmitate concentration-dependently attenuated acetylcholine-induced endothelium-dependent relaxations, up-regulated the expression of cyclooxygenase-2 (COX-2) and elevated superoxide formation in mouse aortic endothelial cells (MAECs) measured by dihydroethidium (DHE) staining and electron paramagnetic resonance (EPR) spectroscopy. Superoxide scavengers, COX-2 inhibitor and thromboxane prostanoid (TP) receptor antagonist, but not COX-1 inhibitor reversed the harmful effects of palmitate. Furthermore, palmitate impaired acetylcholine-induced relaxations and raised superoxide in en face endothelium of aortas only from COX-1(-/-) mice but not from COX-2(-/-) mice. Palmitate increased the production and release of TXB2, a stable thromboxane A2 metabolite in mouse aortas, which was abolished by COX-2 inhibitor. Superoxide scavenger did not affect palmitate-induced up-regulated expression of COX-2 in MAECs. Both real time PCR and luciferase reporter gene assay confirmed COX-2 up-regulation in palmitate-treated MAECs and NF-κB was substantially involved in this up-regulation. The present study provides novel evidence that palmitate up-regulates COX-2 through NF-κB-dependent mechanism and resultant COX-2-associated oxidative stress impairs endothelium-dependent relaxations in mouse aortas.

Tissue-specific metabolite profiling of Cyperus rotundus L. rhizomes and (+)-nootkatone quantitation by laser microdissection, ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and gas chromatography-mass spectrometry techniques.

Cyperus rotundus L. is a plant species commonly found in both India and China. The caused destruction of this plant is of critical concern for agricultural produce. Nevertheless, it can serve as a potential source of the commercially important sesquiterpenoid (+)-nootkatone. The present work describes comparative metabolite profiling and (+)-nootkatone content determination in rhizome samples collected from these two countries. Laser dissected tissues, namely, the cortex, hypodermal fiber bundles, endodermis, amphivasal vascular bundles, and whole rhizomes were analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Gas chromatography-mass spectrometry (GC-MS) analysis was used for profiling of essential oil constituents and quantitation of (+)-nootkatone. The content of (+)-nootkatone was found to be higher in samples from India (30.47 µg/10 g) compared to samples from China (21.72 µg/10 g). The method was validated as per International Conference on Harmonisation (ICH) guidelines (Q2 R1). The results from this study can be applied for quality control and efficient utilization of this terpenoid-rich plant for several applications in food-based industries.

Mass spectrometric studies on effects of counter ions of TMPyP4 on binding to human telomeric DNA and RNA G-quadruplexes.

A comparative study on human telomeric DNA G-quadruplex binding of meso-5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) between its two salt forms, i.e., tetratosylate and tetrachloride, was conducted by using ESI-TOF-MS, UV-melting measurement, and molecular modeling methods. Besides cation TMPyP4, the tosyl anion was found to bind to human telomeric DNA G-quadruplex with multiple binding stoichiometries from 1:1 to 3:1 observed in ESI-TOF-MS spectra, indicating that the stabilization activity of TMPyP4 tetratosylate on G-quadruplex is derived from a synergetic effect of both TMPyP4 cation and tosyl anion. A molecular modeling study suggests that a tosyl anion fills up the vacant space between TMPyP4 cation and DNA G-quadruplex and thus stabilizes the complex by 3.8 kcal/mol. Therefore, it is estimated that TMPyP4 tetratosylate's activity might not reflect the real effect of TMPyP4 cation in some bioassays related to G-quadruplex stabilization. This was verified by the results of less binding affinity of TMPyP4 tetrachloride with DNA G-quadruplex obtained from ESI-TOF-MS measurement, and of 2.27 °C less thermal stabilization of TMPyP4 tetrachloride for DNA G-quadruplex, compared to its tetratosylate under the same conditions. Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding, which sheds light on the proper usage of TMPyP4 salt in the chemical and biological research associated with G-quadruplex binding. Subsequently, the binding of TMPyP4 tetrachloride to human telomeric RNA G-quadruplexes was studied with ESI-TOF-MS technique. The binding constants of TMPyP4 with human telomeric G-quadruplexes indicated that TMPyP4 binds to human telomeric RNA G-quadruplex one order of magnitude stronger than DNA counterpart. This is a comprehensive mass spectrometric report on binding study of TMPyP4 with human telomeric DNA/RNA G-quadruplexes.

Structural diversity requires individual optimization of ethanol concentration in polysaccharide precipitation.

Ethanol precipitation is one of the most widely used methods for preparing natural polysaccharides, in which ethanol concentration significantly affects the precipitate yield, however, is usually set at 70-80%. Whether the standardization of ethanol concentration is appropriate has not been investigated. In the present study, the precipitation yields produced in varied ethanol concentrations (10-90%) were qualitatively and quantitatively evaluated by HPGPC (high-performance gel-permeation chromatography), using two series of standard glucans, namely dextrans and pullulans, as reference samples, and then eight natural samples. The results indicated that the response of a polysaccharide's chemical structure, with diversity in structural features and molecular sizes, to ethanol concentration is the decisive factor in precipitation of these glucans. Polysaccharides with different structural features, even though they have similar molecular weights, exhibit significantly different precipitation behaviors. For a specific glucan, the lower its molecular size, the higher the ethanol concentration needed for complete precipitation. The precipitate yield varied from 10% to 100% in 80% ethanol as the molecular size increased from 1kDa to 270kDa. This paper aims to draw scientists' attention to the fact that, in extracting natural polysaccharides by ethanol precipitation, the ethanol concentration must be individually optimized for each type of material.

Transformation of ginsenosides from notoginseng by artificial gastric juice can increase cytotoxicity toward cancer cells.

Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides.

Quality evaluation of commercial Huang-Lian-Jie-Du-Tang based on simultaneous determination of fourteen major chemical constituents using high performance liquid chromatography.

Huang-Lian-Jie-Du-Tang (HLJDT), comprising Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex and Gardeniae Fructus, is one of the commonly used Chinese medicine formulas for clearing heat and detoxifying. Quality control of the herbal complex like Chinese medicine formulas still remains a challenge. The successful approval of botanical drug Veregen by FDA indicated the importance of quantitative analysis in quality control of herbal medicines. In this study, an effective quantitative method based on conventional HPLC-DAD was developed for simultaneous determination of fourteen major ingredients (seven alkaloids, four flavonoids, three terpenes) in HLJDT. The established method was well validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to quality evaluation of commercial HLJDT samples. The developed method can quantitatively determine up to 70% of the chemicals of commercial HLJDT sample and effectively revealed the significant variation in the quality of the commercial HLJDT samples collected from different locations.

Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18) of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC) (14) as well as natural epigallocatechin (EGC, 6). The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

A cellular lipidomic study on the Aß-induced neurotoxicity and neuroprotective effects of EGCG by using UPLC/MS-based glycerolipids profiling and multivariate analysis.

The aim of this study was to investigate the cellular lipid metabolism associated with ß-amyloid peptide (Aß)-induced neurotoxicity as well as the neuroprotective effect of (-)-epigallocatechin gallate (EGCG), a major polyphenol in green tea. An ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS)-based lipidomic approach was developed to screen and identify changes of the glycerolipids (GL) upon Aß treatment with or without the presence of EGCG in PC12 cells. Principle component analysis (PCA) showed that the Aß-treated group was well separated from the control group, whereas the EGCG group was closer to the control group. The GL levels were significantly elevated in Aß-treated cells compared with the control group, but were restored near to normal levels after EGCG treatment. The elevated phosphatidylcholines (PCs) levels observed in the Aß-treated PC12 cells were quite probably the integrated results of the reduced phospholipase A(2) (PLA(2)) activity and the enhanced activity of lysophospholipid acyltransferases. Moreover, an increased liberation of arachidonic acid (AA) from PCs was observed as another important response of PC12 cells to the Aß aggregates, implying an active inflammatory process occurring in Aß induced neurotoxicity. EGCG treatment can reverse the deregulated metabolism of PCs, which might be one of the biochemical mechanisms contributing to its neuroprotective effect. Collectively, results obtained from the current lipidomic analyses of PC12 cells provided important insight into the biochemical mechanisms underlying Aß-induced neurotoxicity and neuro protective effects of EGCG. This is the first report of the lipidomic study on the neuroprotective effect of EGCG.

Paeoniflorin reduced acute toxicity of aconitine in rats is associated with the pharmacokinetic alteration of aconitine.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the influence of paeoniflorin (major bioactive component of Paeonia lactiflora Pall.) on the pharmacokinetic behavior of aconitine (major toxic and bioactive component of Aconitum carmichaeli Debx.) and potential detoxifying effect of paeoniflorin on the acute toxicity of aconitine, which may provide in depth understanding to the toxicity reduction effect of Paeonia lactiflora to Aconitum carmichaeli. MATERIALS AND METHODS: Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was employed to determine the plasma content of aconitine. Aconitine was administrated by oral to SD rats at the dosage of 200 µg/kg with or without paeoniflorin given by intraperitoneal injection at the dosage of 20 mg/kg. Plasma samples were collected for determination and analysis of pharmacokinetic parameters of aconitine. The LD(50) of aconitine and acute animal death induced by aconitine were examined when aconitine was given alone or jointly with paeoniflorin in ICR mice. RESULTS: A sensitive, accurate, precise, reliable and repeatable UHPLC-MS/MS method was successfully established for determination of the plasma content of aconitine in 12.5 µL plasma sample. The lower limit of quantification of aconitine was 0.01 ng/mL. Compared with the SD rats that were orally administrated with aconitine alone, the rats received aconitine and co-administrated with paeoniflorin by peritoneal injection showed a remarkably lower C(max) (5.69 ng/mL vs 9.66 ng/mL, P<0.05) and delayed T(max) (70 min vs 46 min, P<0.05), as well as a trend of reduction in AUC(0-t) (1082.75 ng-min/mL vs 1650.27 ng-min/mL, P=0.395). The LD(50) values of aconitine coadministered with 120 or 240 mg/kg of paeoniflorin were obviously increased to 2.30 and 2.15 mg/kg against 1.80 mg/kg of aconitine by oral administration alone. Mice treated with paeoniflorin (240 mg/kg) and aconitine (1.8 mg/kg) together revealed a significant decreased death rate than that received aconitine treatment alone (15% vs 50%, P<0.05). CONCLUSIONS: The acute oral toxicity of aconitine in rats was significantly reduced by paeoniflorin; this might result from the alterations of pharmacokinetic behavior of aconitine in the animals by coadministration of paeoniflorin.

Quantitative comparison of ginsenosides and polyacetylenes in wild and cultivated American ginseng.

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg(1)/Rd ratio presented a significantly large difference between cultivated and type-I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg(1)+Re)/Rd, and the ratio of protopanaxatriol (PPT)-type ginsenosides to protopanaxadiol (PPD)-type ginsenosides showed a large difference between these two groups. On the other hand, type-II wild samples were found to have high Rg(1)/Rb(1) and Rg(1)/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type-I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type-II wild American ginseng may have distinct pharmacological activities and therapeutic effects.