Dispersive Liquid-Liquid Microextraction Combined with Microwave Demulsification for Determination of FAME Residuals in Biodiesel Wastewater.

Biodiesel consists of various fatty acid methyl esters (FAMEs) that are mainly produced through transesterification of plant oil or animal fat. It is essential for biodiesel to be purified utmostly to meet its product standard before being traded, while the universal purification method has been water washing. However, water washing inevitably causes the residual of FAMEs in wastewater, which represents a loss of industrial profits. For the purpose of determination and monitoring of the FAME profile in wastewater, there is a necessity to develop a fast and reliable approach with small volume of sample in need. Hence, in this study, a combination of dispersive liquid-liquid microextraction (DLLME) and microwave demulsification is applied for the enrichment of residual FAMEs in water, followed by qualitative and quantitative analyses using gas chromatography-mass spectrometry. The results indicate that the optimal extractant in DLLME approach is toluene. And the optimal parameters are 20 mL of water sample, 80 µL of toluene as the extractant, 60 s of ultrasonic irradiation duration, 200 W of microwave power and 2 min of microwave irradiation duration. The standard curves and linear equations obtained with these conditions are used for the quantitative analysis of biodiesel wastewater, which reveals that there was 50.35 mg·L-1 of the total FAME residuals in wastewater. To the best of our knowledge, it is for the first time that the combined technique of DLLME and microwave demulsification is applied in determination of residual FAMEs in water samples. The proposed method corresponds to small volumes of sample and extractant and short analytical period. It also has the potential to be extended to the analysis of other water pollutants.

Aromatic monoterpenoid glycosides from rattan stems of Schisandra chinensis and their neuroprotective activities.

Six new monoterpenoid glycosides, including thymoquinol 5-O-α-L-arabinopyranosyl-(1 → 6)-ß-D-glucopyranoside (1), cuminic acid 7-O-α-D-arabinofuranosyl-(1 → 6)-ß-d-glucopyranosyl ester (2), p-cymene 7-O-α-D-arabinofuranosyl-(1 → 6)-ß-D-glucopyranoside (3), p-methylhyd- ratropic acid 9-O-α-D-arabinofuranosyl-(1 → 6)-ß-d-glucopyranosyl ester (4), (R)-p-cymene 9-O-α- D-arabinofuranosyl-(1 → 6)-ß-D-glucopyranoside (5), and (R)-p-cymene 9-O-ß-D-apiofuranosyl-(1 → 6)-ß-D-glucopyranoside (6), together with three known compounds, such as (R)-p-cymene 9-O-ß-D-glucopyranoside (7), thymoquinol 5-O-ß-D-glucopyranoside (8), and thymoquinol 2-O-ß-D-glucopyranoside (9) were isolated from the 70%-EtOH extract of Schisandra chinensis rattan stems. The structures of the new compounds were elucidated by detailed spectroscopic analyses. Meanwhile, the neuroprotective activities of compounds 1-9 were evaluated on the two co-culture models of microglia and neurons cells and astrocytoma and neurons cells.

Lysophosphatidic Acid Analogue rather than Lysophosphatidic Acid Promoted the Bone Formation In Vivo.

Lysophosphatidic acid (LPA), a bioactive lipid molecule, has recently emerged as physiological and pathophysiological regulator in skeletal biology. Here we evaluate the effects of LPA on bone formation in vivo in murine femoral critical defect model. Primary femoral osteoblasts were isolated and treated with osteogenic induction conditional media supplemented with 20 µM LPA or LPA analogue. Mineralized nodules were visualized by Alizarin Red S staining. Forty-five C57BL/6 mice underwent unilateral osteotomy. The femoral osteotomy gap was filled with porous scaffolds of degradable chitosan/beta-tricalcium phosphate containing PBS, LPA, or LPA analogue. 2, 5, and 10 weeks after surgery, mice were sacrificed and femurs were harvested and prepared for Micro-Computed Tomography (Micro-CT) and histological analysis. Alizarin Red S staining showed that LPA and LPA analogue significantly enhanced the mineral deposition in osteoblasts. Micro-CT 3D reconstruction images and HE staining revealed that significantly more newly formed bone in osteotomy was treated with LPA analogue when compared to control and LPA group, which was verified by histological analysis and biomechanical characterization testing. In summary, our study demonstrated that although LPA promotes mineralized matrix formation in vitro, the locally administrated LPA was not effective in promoting bone formation in vivo. And bone formation was enhanced by LPA analogue, administrated locally in vivo. LPA analogue was a potent stimulating factor for bone formation in vivo due to its excellent stability.

Characterization of the Isosteroidal Alkaloid Chuanbeinone from Bulbus of Fritillaria pallidiflora as Novel Antitumor Agent In Vitro and In Vivo.

Bulbus of Fritillaria pallidiflora, the dried bulb of F. pallidiflora, is widely used in Chinese folk medicine due to its powerful biological activities. The aim of this study was to investigate in vitro and in vivo antitumor activity of different fractions and isosteroidal alkaloids from bulbus of F. pallidiflora and clarify its putative mechanism of antitumor activity. Firstly, we assayed in vitro antitumor effects of different fractions from bulbus of F. pallidiflora and found that chloroform extracts and purified total alkaloids of bulbus of F. pallidiflora showed higher cytotoxic activity than other tested extracts. We further isolated four main alkaloids, chuanbeinone, imperialine-ß-N-oxide, isoverticine and isoverticine-ß-N-oxide, from the total alkaloids of bulbus of F. pallidiflora and found that they display significant cytotoxicity, whereby chuanbeinone showed the highest activity against Lewis lung carcinoma cells. Moreover, we found that chuanbeinone induced S phase arrest and further increased apoptosis of Lewis lung carcinoma cells. The results of Western blotting experiments showed that the expression of the antiapoptotic Bcl-2 was reduced by chuanbeinone treatment, while the proapoptotic protein Bax and caspase-3 were increased. Moreover, we investigated the in vivo antitumor activity of chuanbeinone and characterized its putative antitumor mechanism of action by the TUNEL assay and by histological and immunohistochemical analyses. Our results showed that chuanbeinone exhibited significant antitumor activity in vivo, while notably inhibiting tumor angiogenesis and inducing apoptosis characterized by an increased expression of caspase-3. Our findings show that chuanbeinone exhibits significant antitumor activity in vitro and in vivo, and points to possible therapeutic potential for this compound as well as for its natural source, bulbus of F. pallidiflora.

[Chemical constituents from EtOAc fraction of Sophora dunnii].

Sixteen compounds have been isolated from the EtOAc fraction of 95% ethanolic extract of Sophora dunnii through silica gel, Sephadex LH-20 and semi-prerarative HPLC column chromatographies. Their structures were identified on the basis of NMR and MS spectra data as phaseollidin (1), L-maackiain (2), 2-(2',4'-dihidroxyphenyl)-5,6-methylenedioxy benzofuran (3), 8-demethyl-farrerol (4), liquiritigenin (5), genistein (6), 6-methylgenistein (7), 5-O-methyl genistein (8), 7,2',4'-trihydroxys-5-methoxy-isoflavanone (9), 7, 3', 4'-trihydroxy-isoflavanone (10), erythribyssin D (11), calycosin (12), trans-resveratrol (13), cis-resveratrol (14), stigmasterol (15), ß-sitosterol (16). Among these, compounds 1-14 and 16 were isolated from this plant for the first time.

[Variability of soil water soluble organic carbon content and its response to temperature change in green spaces along urban-to-rural gradient of Nanchang, China].

Topsoil of green space including typical forest, shrub and grassland were collected to measure their water soluble organic carbon ( WSOC) before and after incubation of 30 days at 5, 15, 25, 35 and, 45 °C. The results showed the average values of WSOC were higher in urban than in rural green spaces, but the percentage of WSOC to total organic carbon (TOC) showed an opposite trend. No significant changes were found among the three green space types in WSOC and WSOC/TOC. Response of WSOC in green space to incubation temperature was generally highest in urban sites, followed by suburban sites, and lowest in rural sites at the incubation temperature of 5 °C, but showed an opposite trend at the temperature of 45 °C. Response coefficient of WSOC to temperature change was lower in forest and shrub than in grassland, but increased along the urban-rural gradient. Further analysis showed that WSOC positively correlated with TOC, total nitrogen and available phosphorus, and the response coefficient of WSOC to temperature change negatively correlated with available phosphorus. In summary, exogenous substances input might lead to the accumulation of WSOC in urban green space, however, urban environment was helpful to maintain the stability of WSOC, which might be due to the enrichment of available phosphorus in urban sites.

In vitro and in vivo antitumor activity of Bulbus Fritillariae Cirrhosae and preliminary investigation of its mechanism.

Bulbus Fritillariae Cirrhosae (BFC) is widely used in China both for food and folk medicine because of its powerful biological activities. Firstly, this study was designed to examine the antiproliferative activities of the different fractions from BFC in vitro by MTT assay. The results showed that chloroform extracts (CE) and the purified total alkaloids of BFC (TAF) exhibited stronger antiproliferative activity than the other fractions. We further determined the total alkaloids and 3 main alkaloids monomers content of CE and TAF by UV and HPLC-ELSD methods, respectively. Moreover, we assessed the antitumor activity of TAF in vivo and made preliminary investigation of its antitumor mechanism by histological and immunohistochemical staining technique. These results demonstrate that TAF showed significant antitumor activity and low toxicity in vivo. Meanwhile, TAF significantly inhibited tumor angiogenesis and induced apoptosis by improvement of expression level of caspase-3. These results suggest that alkaloids of BFC could hold a good potential for use as an antitumor drug.

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action. MATERIALS AND METHODS: The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice. RESULTS: Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo. CONCLUSION: This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.

Effects of acupuncture on Chinese medicine syndromes of vascular dementia.

OBJECTIVE: To study the effects of acupuncture on Chinese medicine syndromes of vascular dementia (VaD). METHODS: Sixty-three VaD patients were divided into three groups. Those willing to be randomized were randomly assigned to receive either acupuncture (random acupuncture group, 24 cases) or rehabilitation training (guided rehabilitation group, 24 cases) for 6 weeks. Those unwilling to be randomized also received acupuncture for 6 weeks (non-random acupuncture group, 19 cases). Patient syndromes and their severity were evaluated before treatment (baseline), at the end of treatment, and at 4-week follow-up after the completion of treatment using a CM scoring system (scale of differentiation of syndromes of vascular dementia, SDSVD). The SDSVD scores of the random and non-random acupuncture groups, and of all patients who received acupuncture (combined acupuncture group, 43 cases), were compared with those in the guided rehabilitation group. RESULTS: In the random, non-random, and combined acupuncture groups, SDSVD scores were significantly reduced at the end of treatment and at follow-up than at baseline. In the guided rehabilitation group, SDSVD scores were similar to baseline scores at the end of treatment and at follow-up. However, there were no significant differences in SDSVD scores among the three groups or between the combined acupuncture group and the guided rehabilitation group at any time points. In the non-random and combined acupuncture groups, SDSVD scores were significantly reduced at the end of treatment than at baseline in patients with hyperactivity of Liver (Gan)-yang or phlegm obstruction of the orifices. CONCLUSIONS: Acupuncture reduced the severity of VaD. The improvement was the greatest in patients undergoing their treatment of choice. Treatments in this study were more effective for excess syndromes, such as Liver-yang hyperactivity or phlegm obstruction of the orifices than deficiency syndromes, such as Kidney (Shen)-essence deficiency.

Effect of acupuncture on hippocampal Ref-1 expression in cerebral multi-infarction rats.

Redox effector factor (Ref-1) is a sensitive marker for oxidative cellular injury. The aim of this study was to investigate the effects of acupuncture on hippocampal Ref-1 expression in cerebral multi-infarction rats. The rats with reference memory impairment were randomly allocated to three groups: impaired group, acupuncture group and placebo acupuncture group. Moreover, normal group and sham-operated group were set as control groups. Morris water maze test showed that cerebral multi-infarction rats did not present significant changes in spatial working memory performance. Further investigation by immunohistochemistry revealed that acupunctural treatment significantly increased the expression of Ref-1 in the hippocampus of the impaired rats. These findings suggested that the spatial working memory was unaffected in the cerebral multi-infarction rats although spatial reference memory deficits were detected in our previous study; in addition, acupuncture could increase the Ref-1 expression, consequently exerting the anti-oxidant effects.

[Phytoplankton' s community structure and its relationships with environmental factors in an aquaculture lake, Datong Lake of China].

From December 2008 to October 2009, a seasonal investigation was conducted on the phytoplankton' s community structure and its relationships with environmental factors in Datong Lake. With the comparison of the historical data in 1960, the potential effects of intensive aquaculture on the aquatic environment were analyzed, aimed to provide theoretical support for the sustainable fishery of freshwater lakes. A total of 98 phytoplankton species belonging to 7 phyla and 54 genera were collected, among which, Peridinium bipes, Chroomonas acuta, Chlorella vulgaris, Crytomonas ovate, Cyclotella meneghiniana, Crytomonas erosa, Anabaena circinalis, Microcystis aeruginosa, and Anabaena azotica were the dominant species, and had obvious seasonal variations. The mean annual cell density of the phytoplankton was 1.84 x 10(6) cells x L(-1), being the highest in summer (16.4 x 10(6) cells x L(-1)) and ranged from 1.71 x 10(6) to 1.98 x 10(6) cells x L(-1) in the other three seasons. The values of the abundance index, Shannon index, and Pielou index of the phytoplankton community were 2.01-4.55, 1.26-2.69, and 0.69-1.27, respectively. Canonical correlation analysis (CCA) showed that water depth, water temperature, transparency, and water total phosphorus content, oxidation-reduction potential, and electrical conductivity were the main environmental factors affecting the phytoplankton community structure in the Lake.

[Progress of researches on acupuncture manipulation and its quantification].

The acupuncture manipulation is an important factor for achieving a good therapeutic effect in acupuncture practice. The authors review recent studies on acupuncture manipulation and its qualification from (a) differences of the therapeutic effects of acupuncture manipulation, (b) quantification of acupuncture manipulations, and (c) instruments for delivering and sampling the analogued parameters of different acupuncture manipulations, computer analysis software, etc. Most studies revealed that different acupuncture manipulations had different clinical efficacies. Meanwhile, researches on the quantification of acupuncture manipulations and some related instruments also have made a bigger progress. Nevertheless,there still exist many problems, such as difficulties of various acupuncture techniques in unification in clinic, stronger personal subjectivity in performing manipulations, etc. We should overcome these problems and conduct more researches, so as to raise the clinical curative effect further.

Quantitative and chemical fingerprint analysis for quality control of rhizoma Coptidischinensis based on UPLC-PAD combined with chemometrics methods.

To control the quality of rhizoma Coptidis, a method based on ultra performance liquid chromatography with photodiode array detector (UPLC-PAD) was developed for quantitative analysis of five active alkaloids and chemical fingerprint analysis. In quantitative analysis, the five alkaloids showed good regression (R > 0.9992) within test ranges and the recovery of the method was in the range of 98.4-100.8%. The limit of detections and quantifications for five alkaloids in PAD were less than 0.07 and 0.22 microg/ml, respectively. In order to compare the UPLC fingerprints between rhizoma Coptidis from different origins, the chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), principal component analysis (PCA) were applied to classify the rhizoma Coptidis samples according to their cultivated origins. Consistent results were obtained to show that rhizoma Coptidis samples could be successfully grouped in accordance with the province of origin. Furthermore, five marker constituents were screened out to be the main chemical marker, which could be applied to accurate discrimination and quality control for rhizoma Coptidis by quantitative analysis. This study revealed that UPLC-PAD method was simple, sensitive and reliable for quantitative and chemical fingerprint analysis, moreover, for the quality evaluation and control of rhizoma Coptidis.

Spectrum-effect relationships between ultra performance liquid chromatography fingerprints and anti-bacterial activities of Rhizoma coptidis.

The fingerprints of Rhizoma coptidis from various sources were established by ultra performance liquid chromatography (UPLC) and the anti-bacterial activities of R. coptidis on Escherichia coli (E. coli) growth was studied by microcalarimetry. The UPLC fingerprints were evaluated using similarity analysis (SA) and hierarchical clustering analysis (HCA). Some quantitative parameters obtained from the thermo-genic curves of E. coli growth affected by R. coptidis were analyzed using principal component analysis (PCA). The spectrum-effect relationships between UPLC fingerprints and anti-bacterial activities were investigated using canonical correlation analysis (CCA). The results showed that close correlation existed between the spectrum-effect relationships. Berberine, jateorrhizine and palmatine in the UPLC fingerprints might be the main anti-bacterial components. The anti-bacterial activities of R. coptidis were related with the main active constituents, along with the production place and the harvesting time of this herb, the latitude and longitude of the place. This work provides a general model of the combination of UPLC and microcalorimetry to study the spectrum-effect relationships of R. coptidis, which can be used to discover principle components of it on bioactivity.

Effect of acupuncture treatment on spastic states of stroke patients.

PURPOSES: The control of spasticity is often a significant problem in the management of patients with stroke. The aim of this study was to evaluate the effect of acupuncture treatment on the spastic states of stroke patients. SETTING: An outpatient Acupuncture Department in the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine. PARTICIPANTS: One hundred and thirty-one patients, mean (SD) age of 59 (12) years, with spastic hemiplegia were included at mean (SD) month of 17 (7) months after stroke. INTERVENTION: Participants received two 30-day treatment regimens: combined stimulating surface projection zone of decussation of pyramid and traditional acupuncture treatment, and traditional acupuncture treatment only. MAIN OUTCOME MEASURES: Differences in the modified Ashworth scale (MAS), Fugl-Meyer Assessment (FMA), Barthel Index (BI), and the electromyographic activity of the affected extremity between arms. RESULTS: The average (+/-SD) upper extremity Ashworth score significantly decreased, from 3.08+/-0.77 before treatment to 1.82+/-0.65 after acupuncture intervention (wrist joint, P<0.05), and from 2.72+/-0.59 to 1.32+/-0.71 (elbow joint, P<0.05) for treatment group. There were significant differences noted between the treatment group and control group after administration. Lower extremity treatment responses were similar to upper extremity responses. However, both groups showed similar improvement in FMA (upper extremity) and FMA (lower extremity). However, the improvements of FMA (total), BI, and F/M ratio were better in treatment group than in control group. CONCLUSIONS: These results suggested that acupuncturing surface projection zone of decussation of pyramid was effective in reducing spastically increased muscle tone and motor neuron excitability in spastic hemiplegia, and could improve spastic states of stroke patients, thus providing a safe and economical method for treating stroke patients.

Quantitative structure-activity relationship studies on 1-aryl-tetrahydroisoquinoline analogs as active anti-HIV agents.

Predictive quantitative structure-activity relationship analysis was developed for a diverse series of recently synthesized 1-aryl-tetrahydroisoquinoline analogs with anti-HIV activities in this study. The conventional 2D-QSAR models were developed by genetic function approximation (GFA) and stepwise multiple linear regression (MLR) with acceptable explanation of 94.9% and 95.5% and good predicted power of 91.7% and 91.7%, respectively. The results of the 2D-QSAR models were further compared with 3D-QSAR model generated by molecular field analysis (MFA), investigating the substitutional requirements for the favorable receptor-drug interaction and quantitatively indicating the important regions of molecules for their activities. The results obtained by combining these methodologies give insights into the key features for designing more potent analogs against HIV.

[Methodological study on quality evaluation of rhubarb based on purgative bioassay].

OBJECTIVE: To establish a method suitable to determine the purgative biopotency of rhubarb and construct a new quality evaluation pattern of rhubarb. METHOD: A series of factors such as observation index (mass of feces in 10 hours), animal strain (ICR mice), sex (male) and the dose of diphenoxylate complex (50 mg x kg(-1)) was investigated and fixed. The purgative biopotency as well as anthraquinone determination was used to evaluate the quality of different rhubarb samples. RESULT: There wasn't a good linear relationship between the purgative biopotency and content of anthraquinone. The quality difference of rhubarb samples could be well characterized by combination of purgative biopotency determination and anthraquinone determination. CONCLUSION: The purgative biopotency determination can be used in quality control and evaluation of rhubarb.

[Effects of 3 genosides on bio-thermodynamic expression of splenic lymphocyte in mice].

OBJECTIVE: To establish a new method for screening active ingredients of Chinese herbs by determining different bio-thermodynamic effects of 3 genosides on splenic lymphocyte of mice. METHODS: Using a thermal bioactivity monitoring system, the maximum heat output (mHO), average metabolic heat (MH) and constant of decrease rate (DR) of lymphocyte were determined based on the growth metabolic power-time curve, and the outcomes were verified by MIT. RESULTS: The mHO and MH increased and the DR decreased after lymphocytes being exposed to the 3 genosides in different concentrations, arranged upon their potency as genoside Rg3 > genoside Rg2 > genoside Rg1 (merely insignificant effect). MTT showed the same results. CONCLUSION: Heat activity monitoring system could precisely display the different bio-thermal dynamic effects of 3 genosides on splenic lymphocyte.

Direct determination of fourteen underivatized amino acids from Whitmania pigra by using liquid chromatography-evaporative light scattering detection.

A new reversed phase high performance liquid chromatography-evaporative light scattering detection (RP-HPLC-ELSD) method has been developed for the direct determination of fourteen amino acids in Whitmania pigra, a commonly used traditional Chinese medicine (TCM). Simultaneous separation of these fourteen amino acids was achieved on a Prevail C18 column. Under the condition of gradient elution, the fourteen amino acids were separated within 25 min. The drift tube temperature of ELSD was set at 115 degrees C, and with the nitrogen flow rate of 2.5 l/min. All calibration curves showed good linear regression (r > 0.9975) within test ranges. The recoveries ranged from 94.8% to 104.4%. The limit of detection (LOD) for each compound was more than 20 mg/l at a signal to noise ratio (S/N) of 3. The validated method was successfully applied to quantify fourteen amino acids from Whitmania pigra, which provides a new basis of overall assessment on quality of Whitmania pigra.

[The determination of contents of 8 ginsenosides in extraction of Panax ginseng by HPLC].

OBJECTIVE: To establish a method to determine the contents of 8 ginsenosides in extraction of Panax ginseng by HPLC. METHODS: The sample was analyzed on an ODS chromatogram column (Kromasil 250 mm x 4.6 mm, 5 microm), with mobile phase of acetonitrile-water (gradient elution) at flow rate 1.0 ml/min and detection at wavelength of 203 nm. RESULTS: RSD of stability, precision and recurrency was 0.55%-2.26%, 0.85%-1.93% and 0.97%-2.72% respectively. CONCLUSION: This method can be good for the content determination of ginsenoside.