A new neuroprotective candidate TJ1 targeting amyloidogenesis in 5xFAD Alzheimer's disease mice.

As one of the main pathmechanisms of Alzheimer's disease (AD), amyloid-ß (Aß) is widely considered to be the prime target for the development of AD therapy. Recently, imidazolylacetophenone oxime ethers or esters (IOEs) have shown neuroprotective effects against neuronal cells damage, suggesting their potential use in the prevention and treatment of AD. Thirty IOEs compounds from our lab in-house library were constructed and screened for the inhibitory effects on Aß42-induced cytotoxicity. Among them, TJ1, as a new IOEs hit, preliminarily showed the effect on inhibiting Aß42-induced cytotoxicity. Furthermore, the inhibitory effects of TJ1 on Aß42 aggregation were tested by ThT assays and TEM. The neuroprotective effects of TJ1 were evaluated in Aß42-stimulated SH-SY5Y cells, LPS-stimulated BV-2 cells, and H2O2- and RSL3-stimulated PC12 cells. The cognitive improvement of TJ1 was assessed in 5xFAD (C57BL/6J) transgenic mouse. These results showed that TJ1 had strong neuroprotective effects and high blood-brain barrier (BBB) permeability without obvious cytotoxicity. TJ1 impeded the self-accumulation process of Aß42 by acting on Aß oligomerization and fibrilization. Besides, TJ1 reversed Aß-, H2O2- and RSL3-induced neuronal cell damage and decreased neuroinflammation. In 5xFAD mice, TJ1 improved cognitive impairment, increased GSH level, reduced the level of Aß42 and Aß plaques, and attenuated the glia reactivation and inflammatory response in the brain,. Taken together, our results demonstrate that TJ1 improves cognitive impairments as a new neuroprotective candidate via targeting amyloidogenesis, which suggests the potential of TJ1 as a treatment for AD.

Acupuncture attenuates cognitive impairments in vascular dementia through inhibiting miR-143-3p.

BACKGROUND: Acupuncture can be used to treat vascular dementia (VD), but the underlying mechanism remains unclear. This study aimed to investigate the expression and clinical significance of microRNA-143-3p (miR-143-3p) in VD patients and explore whether acupuncture ameliorates VD by regulating miR-143-3p. METHODS: Cognitive function and daily living ability in VD patients were assessed by mini-mental state examination, Hasegawa's dementia scale and activities of daily living scale, respectively. VD model of male Wistar rats was established using permanent bilateral common carotid artery occlusion. The expression level of miR-143-3p was measured by quantitative real-time PCR. Morris water maze test was used to assess the cognitive function of VD rat model. Receiver operating characteristic analysis was used to assess the diagnostic value of miR-143-3p in VD patients. Correlations between variables were analyzed by Pearson's correlation analysis. RESULTS: Increased serum miR-143-3p expression in VD patients had a high diagnostic value to screen VD patients. Serum miR-143-3p level in VD patients after acupuncture treatment was decreased. After acupuncture treatment, serum miR-143-3p was negatively correlated with cognitive function and daily living ability in VD patients. miR-143-3p level was increased in VD rats, and the suppressive effects of acupuncture on miR-143-3p levels were relieved by miR-143-3p mimic. Overexpression of miR-143-3p reversed the ameliorative effect of acupuncture on cognitive functions of VD rats. CONCLUSION: Serum miR-143-3p expression is upregulated in VD patients and downregulated in VD patients after acupuncture treatment. Additionally, acupuncture treatment may attenuate cognitive impairments in VD by suppressing miR-143-3p.

Artificial Intelligence in Coronary CT Angiography: Current Status and Future Prospects.

Coronary heart disease (CHD) is the leading cause of mortality in the world. Early detection and treatment of CHD are crucial. Currently, coronary CT angiography (CCTA) has been the prior choice for CHD screening and diagnosis, but it cannot meet the clinical needs in terms of examination quality, the accuracy of reporting, and the accuracy of prognosis analysis. In recent years, artificial intelligence (AI) has developed rapidly in the field of medicine; it played a key role in auxiliary diagnosis, disease mechanism analysis, and prognosis assessment, including a series of studies related to CHD. In this article, the application and research status of AI in CCTA were summarized and the prospects of this field were also described.

Diagnostic Potential of Plasma Extracellular Vesicle miR-483-3p and Let-7d-3p for Sepsis.

Background: microRNAs (miRNAs) from circulating extracellular vesicles (EVs) have been reported as disease biomarkers. This study aimed to identify the diagnostic value of plasma EV-miRNAs in sepsis. Methods: EVs were separated from the plasma of sepsis patients at admission and healthy controls. The expression of EV-miRNAs was evaluated by microarray and qRT-PCR. Results: A preliminary miRNA microarray of plasma EVs from a discovery cohort of 3 sepsis patients at admission and three healthy controls identified 11 miRNAs with over 2-fold upregulation in sepsis group. Based on this finding, EV samples from a validation cohort of 37 sepsis patients at admission and 25 healthy controls were evaluated for the expression of the 6 miRNAs relating injury and inflammation via qRT-PCR. Elevated expression of miR-483-3p and let-7d-3p was validated in sepsis patients and corroborated in a mouse model of sepsis. miR-483-3p and let-7d-3p levels positively correlated with the disease severity. Additionally, a combination of miR-483-3p and let-7d-3p had diagnostic value for sepsis. Furthermore, bioinformatic analysis and experimental validation showed that miR-483-3p and let-7d-3p target pathways regulating immune response and endothelial function. Conclusion: The present study reveals the potential role of plasma EV-miRNAs in the pathogenesis of sepsis and the utility of combining miR-483-3p and let-7d-3p as biomarkers for early sepsis diagnosis.

Cognitive enhancement and neuroprotective effects of OABL, a sesquiterpene lactone in 5xFAD Alzheimer's disease mice model.

Alzheimer's disease (AD) is a neurodegenerative disease in which oxidative stress and neuroinflammation were demonstrated to be associated with neuronal loss and cognitive deficits. However, there are still no specific treatments that can prevent the progression of AD. In this study, a screening of anti-inflammatory hits from 4207 natural compounds of two different molecular libraries indicated 1,6-O,O-diacetylbritannilactone (OABL), a 1,10-seco-eudesmane sesquiterpene lactone isolated from the herb Inula britannica L., exhibited strong anti-inflammatory activity in vitro as well as favorable BBB penetration property. OABL reduced LPS-induced neuroinflammation in BV-2 microglial cells as assessed by effects on the levels of inflammatory mediators including NO, PGE2, TNF-α, iNOS, and COX-2, as well as the translocation of NF-κB. Besides, OABL also exhibited pronounced neuroprotective effects against oxytosis and ferroptosis in the rat pheochromocytoma PC12 cell line. For in vivo research, OABL (20 mg/kg B.W., i.p.) for 21 d attenuated the impairments in cognitive function observed in 6-month-old 5xFAD mice, as assessed with the Morris water maze test. OABL restored neuronal damage and postsynaptic density protein 95 (PSD95) expression in the hippocampus. OABL also significantly reduced the accumulation of amyloid plaques, the Aß expression, the phosphorylation of Tau protein, and the expression of BACE1 in AD mice brain. In addition, OABL attenuated the overactivation of microglia and astrocytes by suppressing the expressions of inflammatory cytokines, and increased glutathione (GSH) and reduced malondialdehyde (MDA) and super oxide dismutase (SOD) levels in the 5xFAD mice brain. In conclusion, these results highlight the beneficial effects of the natural product OABL as a novel treatment with potential application for drug discovery in AD due to its pharmacological profile.

Imidazolylacetophenone oxime-based multifunctional neuroprotective agents: Discovery and structure-activity relationships.

Alzheimer's disease (AD) possesses a complex pathogenetic mechanism. Nowadays, multitarget agents are considered to have potential in effectively treating AD via triggering molecules in functionally complementary pathways at the same time. Here, based on the screening (∼1400 compounds) against neuroinflammation, an imidazolylacetophenone oxime ether (IOE) was discovered as a novel hit. In order to obtain SARs, a series of imidazolylacetophenone oxime derivatives were constructed, and their C=N bonds were confirmed as the Z configuration by single crystals. These derivatives exhibited potential multifunctional neuroprotective effects including anti-neuroinflammatory, antioxidative damage, metal-chelating, inhibition of acetylcholinesterase (AChE) properties. Among these derivatives, compound 12i displayed the most potent inhibitory activity against nitric oxide (NO) production with EC50 value of 0.57 µM 12i can dose-dependently suppress the expression of iNOS and COX-2 but not change the expression of HO-1 protein. Moreover, 12i exhibited evidently neuroprotective effects on H2O2-induced PC12 cells damage and ferroptosis without cytotoxicity at 10 µM, as well as selectively metal chelating properties via chelating Cu2+. In addition, 12i showed a mixed-type inhibitory effect on AChE in vitro. The structure-activity relationships (SARs) analysis indicated that dioxolane groups on benzene ring and rigid oxime ester can improve the activity. Parallel artificial membrane permeation assay (PAMPA) also verified that 12i can overcome the blood-brain barrier (BBB). Overall, this is the first report on imidazolylacetophenone oxime-based multifunctional neuroprotective effects, suggesting that this type of compounds might be novel multifunctional agents against AD.

Identification of NLRP3 as a covalent target of 1,6-O,O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP).

Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative diseases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula britannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear. Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-κB transcription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the α-methylene-γ-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new covalent target of 1 and 2 against neuroinflammation.

1,10-Seco-Eudesmane sesquiterpenoids as a new type of anti-neuroinflammatory agents by suppressing TLR4/NF-κB/MAPK pathways.

Dysregulation of neuroinflammation is a key pathological factor in the progressive neuronal damage of neurodegenerative diseases. An in-house natural products library of 1407 compounds were screened against neuroinflammation in lipopolysaccharide (LPS)-activated microglia cells to identify a novel hit 1,6-O,O-diacetylbritannilactone (OABL) with anti-neuroinflammatory activity. Furthermore, a 1,10-seco-eudesmane sesquiterpenoid library containing 33 compounds was constructed by semisynthesis of a major component 1-O-acetylbritannilactone (ABL) from the traditional Chinese medicinal herb Inula Britannica L. Compound 15 was identified as a promising anti-neuroinflammatory agent by nitrite oxide (NO) production screening. 15 could attenuate tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) productions, and inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at a submicromolar level. Mechanistic study revealed that 15 significantly modulated TLR4/NF-kB and p38 MAPK pathways, and upregulated the anti-oxidant response HO-1. Besides, 15 promoted the conversion of the microglia from M1 to M2 phenotype by increasing levels of arginase-1 and IL-10. The structure-activity relationships (SARs) analysis indicated that the α-methylene-γ-lactone motifs, epoxidation of C5=C10 bond and bromination of C14 were important to the activity. Parallel artificial membrane permeation assay (PAMPA) also demonstrated that 15 and OABL can overcome the blood-brain barrier (BBB). In all, compound 15 is a promising anti-neuroinflammatory lead with potent anti-inflammatory effects via the blockage of TLR4/NF-κB/MAPK pathways, favorable BBB penetration property, and low cytotoxicity.

The prognostic value of geriatric nutritional risk index in elderly patients with severe community-acquired pneumonia: A retrospective study.

The geriatric nutritional risk index (GNRI) is associated with the prognosis of many diseases. However, the association between the GNRI and the prognosis of patients aged ≥65 years with severe community-acquired pneumonia (SCAP) has not been studied. We aimed to evaluate the prognostic value of GNRI in elderly SCAP patients.This study retrospectively analyzed the clinical data of 346 patients aged ≥65 years with SCAP from December 2013 to September 2019. Patients were divided into 4 groups by the GNRI. The chi-square test or student's t test was used to compare the differences between the groups. Logistic regression analysis was used to evaluate the factors that affect prognosis. The receiver operating characteristic curve was used to compare the prognostic performance of the GNRI with other indicators. A GNRI-based nomogram was established based on the result of the multivariate analysis.Two hundred nine (60.4%) patients had a poor prognosis. GNRI scores were significantly lower in the poor prognosis group than in the group with a good prognosis. In the multivariate analysis, gender, mean arterial pressure, neutrophil counts, and the GNRI were independently correlated with the prognosis of elderly patients. The GNRI was a significantly better predictor for poor prognosis than other indicators. The GNRI-based nomogram had excellent prediction capabilities.GNRI is a simple and effective prognostic indicator for elderly patients with SCAP, and a GNRI-based nomogram can aid in developing individualized treatment plans for elderly patients with SCAP.

Human adipose-derived mesenchymal stem cells alleviate obliterative bronchiolitis in a murine model via IDO.

BACKGROUND: Long-term survival of lung transplantation is hindered by the development of obliterative bronchiolitis (OB). Adipose-derived stem cells (ASCs) were documented to have more potent immunosuppressive ability than mesenchymal stem cells (MSCs) from bone marrow and placenta. The goal of our study is to evaluate the effect of repeated administration of ASCs on OB and the involvement of indoleamine 2,3-dioxygenase (IDO) mediating the protective effect of ASCs in a heterotopic tracheal transplantation (HTT) model. METHODS: For studies in vitro, ASCs were treated with interferon-γ (IFN-γ). For in vivo study, tracheas from BALB/c or C57BL/6 donors were transplanted into C57BL/6 recipients to create a HTT model. On days 0, 1, 3, 5, 8, 12, 15, 20 and 25 post-transplant, the allogeneic recipient mice were administered intravenously with phosphate buffered saline, 1 × 106 human ASCs, or 1 × 106 human ASCs plus 1-methyltryptophan (1-MT), an IDO inhibitor. On days 3, 7, 14 and 28, serum, trachea and spleen samples were harvested for analysis. RESULTS: ASCs homed to heterotopic tracheal grafts after infusion. Multiple doses of ASCs significantly increased tracheal IDO levels in allografts. There were significant increases in graft and serum IFN-γ levels in allografts compared with isografts. IFN-γ elevated IDO expression and activity in ASCs in vitro. ASCs alleviated OB in allografts as evidenced by reduced epithelial loss, epithelial apoptosis, and intraluminal obstruction. The effects of ASCs on OB were blocked by 1-MT. 1-MT also blocked the alterations in pro and anti-inflammatory cytokines as well as CD3+ T cell infiltration induced by ASCs. ASCs induced not only splenic levels of CD4+CD25+Foxp3+ regulatory T cells (Treg) but also IL-10 and TGF-ß-producing Treg. Furthermore, IDO inhibition abolished the changes of splenic Treg induced by ASCs. In addition, Treg reduction by cyclophosphamide treatment did not alter the effects of ASCs on tracheal IDO expression in allografts confirming Treg induction is downstream of IDO. CONCLUSIONS: Repeated doses of ASCs are capable of ameliorating OB. ASCs act at least in part via elevating IDO expression. ASCs promote the generation of Treg and suppress T cell infiltration via an IDO-dependent mechanism.

Adipose-derived mesenchymal stem cells modulate CD14++CD16+ expression on monocytes from sepsis patients in vitro via prostaglandin E2.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to reduce sepsis-induced inflammation and improve survival in mouse models of sepsis. CD16+ monocytes are proinflammatory and abundant in inflammatory conditions such as sepsis. The primary objective in this exploratory study was to determine the effects of adipose-derived MSCs (ASCs) on three subsets of monocytes from sepsis patients in vitro and to delineate the underlying mechanism. METHODS: This is a prospective cohort study of patients admitted to the medical intensive care unit (ICU) at an academic medical center. The levels of CD14++CD16+, CD14+CD16++, and CD14++CD16- monocytes from 23 patients in the early phase of severe sepsis or septic shock as well as 25 healthy volunteers were determined via flow cytometry after coculture with or without ASCs. To determine the molecular mechanisms, the effects of exogenous prostaglandin E2 (PGE2) and the cyclooxygenase-2 (COX-2) inhibitor NS-398 on monocyte phenotypes and cytokine expression were also examined. RESULTS: Basal levels of CD14++CD16+ but not CD14+CD16++ monocytes were significantly elevated in severe sepsis and septic shock. A positive linear relationship existed between the levels of CD14++CD16+ monocytes and the Acute Physiology and Chronic Health Evaluation (APACHE) II score as well as Sequential Organ Failure Assessment (SOFA) score. Coculture of ASCs with monocytes from sepsis patients for 24 h significantly reduced CD14++CD16+ expression while increasing the CD14++CD16- phenotype. The coculture also significantly elevated PGE2, COX-2, and prostaglandin E2 receptor (EP)4 levels generated from monocytes. Functionally, ASCs reduced the tumor necrosis factor (TNF)-α and increased the interleukin (IL)-10 secretion in monocytes of septic patients. Furthermore, the effects of ASCs on the CD14++CD16+ phenotype and cytokine expression were mimicked by exogenous PGE2 and abolished by the COX-2 inhibitor NS-398. Additionally, ASCs also modified levels of monocyte phenotypes in a mouse model of sepsis. CONCLUSIONS: Levels of CD14++CD16+ monocytes positively correlate with disease severity scores in the early phase of severe sepsis and septic shock. ASCs switch monocytes of sepsis patients from CD14++CD16+ to CD14++CD16- in vitro and modulate the production of inflammatory cytokines. The immunomodulatory effect of ASCs on monocytes is PGE2-dependent. ASCs may exert their therapeutic effect on sepsis via altering monocyte phenotypes and functions.

Mesenchymal Stem Cell-Educated Macrophages Ameliorate LPS-Induced Systemic Response.

Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory effects. The goal of this study was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model. Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups: control, LPS, LPS + ASCs, LPS + untreated macrophages, and LPS + educated macrophages. Educated macrophages decreased lung inflammation, weight loss, pulmonary edema, and inflammatory cytokine response. In vitro, ASCs increased expression of M2 macrophages independent of direct cell-to-cell contact when macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). When macrophages were cultured with serum from ARDS patients who were treated with ASCs or placebo in our previous clinical trial, there was no difference in M2 macrophage levels before and after ASCs treatment indicating a suboptimal response to the treatment protocol. ASCs also reduced the levels of LPS-induced proinflammatory cytokines in vitro which were mimicked by IL-10 and blocked by antibodies for IL-10 and IL-10 receptor supporting the notion that educated macrophages exert their anti-inflammatory effects via IL-10-dependent mechanisms.

Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study.

BACKGROUND: Recent studies have demonstrated that mesenchymal stem cells (MSCs) modulate the immune response and reduce lung injury in animal models. Currently, no clinical studies of the effects of MSCs in acute respiratory distress syndrome (ARDS) exist. The objectives of this study were first to examine the possible adverse events after systemic administration of allogeneic adipose-derived MSCs in ARDS patients and second to determine potential efficacy of MSCs on ARDS. METHODS: Twelve adult patients meeting the Berlin definition of acute respiratory distress syndrome with a PaO2/FiO2 ratio of < 200 were randomized to receive allogeneic adipose-derived MSCs or placebo in a 1:1 fashion. Patients received one intravenous dose of 1 × 106 cells/kg of body weight or saline. Possible side effects were monitored after treatment. Acute lung injury biomarkers, including IL-6, IL-8 and surfactant protein D (SP-D), were examined to determine the effects of MSCs on lung injury and inflammation. RESULTS: There were no infusion toxicities or serious adverse events related to MSCs administration and there were no significant differences in the overall number of adverse events between the two groups. Length of hospital stay, ventilator-free days and ICU-free days at day 28 after treatment were similar. There were no changes in biomarkers examined in the placebo group. In the MSCs group, serum SP-D levels at day 5 were significantly lower than those at day 0 (p = 0.027) while the changes in IL-8 levels were not significant. The IL-6 levels at day 5 showed a trend towards lower levels as compared with day 0, but this trend was not statistically significant (p = 0.06). CONCLUSIONS: Administration of allogeneic adipose-derived MSCs appears to be safe and feasible in the treatment of ARDS. However, the clinical effect with the doses of MSCs used is weak, and further optimization of this strategy will probably be required to reach the goal of reduced alveolar epithelial injury in ARDS. TRIAL REGISTRATION: Clinical trials.gov, NCT01902082.

Comparative Analysis of the Volatile Components of Agrimonia eupatoria from Leaves and Roots by Gas Chromatography-Mass Spectrometry and Multivariate Curve Resolution.

Gas chromatography-mass spectrometry and multivariate curve resolution were applied to the differential analysis of the volatile components in Agrimonia eupatoria specimens from different plant parts. After extracted with water distillation method, the volatile components in Agrimonia eupatoria from leaves and roots were detected by GC-MS. Then the qualitative and quantitative analysis of the volatile components in the main root of Agrimonia eupatoria was completed with the help of subwindow factor analysis resolving two-dimensional original data into mass spectra and chromatograms. 68 of 87 separated constituents in the total ion chromatogram of the volatile components were identified and quantified, accounting for about 87.03% of the total content. Then, the common peaks in leaf were extracted with orthogonal projection resolution method. Among the components determined, there were 52 components coexisting in the studied samples although the relative content of each component showed difference to some extent. The results showed a fair consistency in their GC-MS fingerprint. It was the first time to apply orthogonal projection method to compare different plant parts of Agrimonia eupatoria, and it reduced the burden of qualitative analysis as well as the subjectivity. The obtained results proved the combined approach powerful for the analysis of complex Agrimonia eupatoria samples. The developed method can be used to further study and quality control of Agrimonia eupatoria.

Study of the volatile constituents in radix flemingiae macrophyllae and a substitute by gas chromatography-mass spectrometry and chemometric methods.

A combined approach of subwindow factor analysis and spectral correlative chromatography was used to analyze the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth, one of its substitutes. After extraction by a water distillation method, the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth were detected by GC-MS. Then the qualitative and quantitative analysis of the volatile components in Radix Flemingiae Macrophyllae was completed with the help of subwindow factor analysis resolving two-dimensional original data into mass spectra and chromatograms. Sixty five of 82 separated constituents in the total ion chromatogram of the volatile components in Radix Flemingiae Macrophyllae were identified and quantified, accounting for about 88.79% of the total content. Then, spectral correlative chromatography was used to extract correlative constituents in Flemingiae Latifolia Benth. Fifty one correlative components were recognized in essential oil of Flemingiae Latifolia Benth. The result proves the combined approach is powerful in the analysis of complex herbal samples. The developed method can be used to compare the sameness and differences of Radix Flemingiae Macrophyllae and its substitutes and it can also be used for quality control of Radix Flemingiae Macrophyllae.

Identification and determination of flavonoids in astragali radix by high performance liquid chromatography coupled with DAD and ESI-MS detection.

A method for the analysis of flavonoids in Astragali Radix by high-performance liquid chromatography (HPLC) combined with photodiode-array detection (DAD) and an electrospray ionization (ESI)--mass spectrometry (MS) was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. Eight flavonoids were identified to exist in Astragali Radix based on their characteristic UV data and mass spectra. The concentrations of three major components in this herb--ononin, calycosin and formononetin--were determined by LC/ESI-MS in positive selective ion monitoring (SIM) mode. The calibration curves were linear in the range of 0.9~180.0 µg·mL⁻¹ for ononin, 1.8~360.0 µg·mL⁻¹ for calycosin and 1.4~280 µg·mL⁻¹ for formononetin, respectively. The limits of quantification (LOQ) and detection (LOD) were 0.9 µg· mL⁻¹ and 0.2 µg mL⁻¹ for ononin, 1.8 µg mL⁻¹ and 0.5 µg·mL-1 for calycosin, 1.4 µg mL⁻¹ and 0.5 µg·mL⁻¹ for formononetin, respectively. The standard recoveries were between 95.4~104.7%. The developed method was proven to be useful for the quantitative and qualitative analysis of flavonoid constituents in various resources of Astragali Radix.

Determination of the volatile constituents in radix Flemingiae Philippinensis by GC-MS and a heuristic evolving latent projection method.

With the help of chemometric resolution methods, a technique for qualitative and quantitative determination of the volatile chemical constituents in radix Flemingiae Philippinensis by chromatography-mass spectrometry was developed. After the overlapping chromatographic peaks were resolved into pure chromatograms and spectra using a heuristic evolving latent projections (HELP) method, qualitative analysis was performed by similarity search of the obtained pure mass spectrum of each component in the NIST library and the quantitative results were obtained by calculating the total two-way response volume. A total of 63 components were separated and 55 components were identified, accounting for 90.62% of the total content. The main components were farnesol isomer and beta-caryophyllene, accounting for 31.33% and 12.60% of the total content, respectively. The obtained results can provide useful information for further study and development of radix Flemingiae Philippinensis.

Analysis of the main nucleosides in Cordyceps sinensis by LC/ESI-MS.

A sensitive, selective and reliable liquid chromatography-mass spectrometry coupled with electrospray ionization interface method for simultaneous separation and determination of thymine, adenine, adenosine and cordycepin in Cordyceps sinensis has been established. The optimum separation for these analytes was achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. 2-Chloroadenosine was used as internal standard for this assay. [M+H]+ ions at m/z 127, 136, 268, 252 and 302 were chosen and selective ion monitoring (SIM) mode was used for quantitative analysis of the four main nucleosides. The regression equations were linear in the range of 1.0-117.5 microg x mL(-1) for thymine, 1.8-127.0 microg x mL(-1) for adenine, 0.6-114.0 microg x mL(-1) for adenosine and 0.5-107.5 microg x mL(-1) for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were 1.0 and 0.2 microg x mL(-1) for thymine, 1.8 and 0.6 microg x mL(-1) for adenine, 0.6 and 0.1 microg x mL(-1) for adenosine and 0.5 and 0.1 microg x mL(-1) for cordycepin, respectively. The recoveries of the four nucleosides ranged from 98.47 to 99.32%. The developed method was successfully used to determine nucleosides in Cordyceps sinensis from different sources.

[Rapid simultaneous determination of main nucleosides in Cordyceps sinensis with LC-ESI-MS].

OBJECTIVE: To establish an LC-MS method for rapid simultaneous determination of adenine,adenosine and cordycepin in Cordyceps sinensis. METHOD: An electrospray ionization (ESI) interface and selective ion monitoring (SIM) mode were used. The analytical column was a Shimadzu VP-ODS column (2.0 mm x 150 mm) and the mobile phase was water-methanol-formic acid (92:7:1). Methanol was used as extraction solvent and 2-chloroadenosine was used as internal standard for this assay. RESULT: The regression equations and coefficient were Y = 0.07264X + 0.00622 and r = 0.9987 for adenine, Y = 0.1597X + 0.0146 and r = 0.9991 for adenosine, Y = 0.1942X + 0.0186 and r = 0.9994 for cordycepin. The linear range was 0.8-130.0 mg x L(-1), 0.5 - 124.5 mg x L(-1) and 0.5-128.5 mg x L(-1) for adenine, adenosine and cordycepin, respectively. The average recovery was 98.76%, 99.37% and 99.26% for adenine, adenosine and cordycepin, respectively. CONCLUSION: This established method was highly sensitive, fast and selective, which can be used for rapid simultaneous determination of adenine, adenosine and cordycepin in C. sinensis. This method also can be applied for the quality control of C. sinensis.

[Headspace solid-phase microextraction-gas chromatography-mass spectrometry for analysis of volatile components from Atractlodes macrocephala Koidz].

Headspace solid-phase microextraction (HS-SPME) technique was employed to extract the volatile components from Chinese traditional medicine, Atractlodes macrocephala Koidz. The volatile components were isolated and identified successfully by gas chromatography-mass spectrometry (GC-MS). The results from HS-SPME-GC-MS were compared with those obtained from steam distillation-GC-MS (SD-GC-MS) with a good agreement. The volatiles were collected using a polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber by HS-SPME. The best response was obtained when the extraction temperature was 70 degrees C, the equilibrium time and extraction time were all 30 min and the desorption time was 4 min. Analysis was performed by GC-MS with a polysiloxane capillary column (30 m x 0.25 mm, film thickness 0.25 microm) using He as the carrier gas and a programmed temperature run. Forty-one components accounting for 90.81% were identified. The main components (relative amount more than 1%) of the samples by HS-SPME were atractylone (40.12%), gamma-elemene (14.73%), aromadendrene (13.05%), eudesma-4 (14), 11-diene (5.46%), caryophyllene (2.56%), patchoulene (2.55%), 6,10,11,11-tetramethyl-tricyclo [6.3.0.1(2,3)] undec-7-ene (2.11%), cedrene (1.48%), alpha-caryophyllene (1.48%) and selina-4 (14) -7 (11) diene-8-one (1.01%). By SD-GC-MS, 31 components accounting for 88.19% were identified and all these components could be extracted by SPME except trans-beta-ocimene which accounts only 0.10%. The results showed that the HS-SPME technique can be used to extract the the volatile components from Atractlodes macrocephala Koidz. in place of the traditional time-consuming SD.