[Quality Evaluation of Saposhnikoviae Radix (Differences between Wild-type and Cultivated Products)].

　Saposhnikoviae Radix ("Boufu") is an important crude drug used in Kampo formulation. It is extracted from wild-type plants. However, recently, extraction has become difficult because of a decrease in wild-type plants. Therefore, cultivated plants account for the majority of the market, from which the crude drug is extracted. However, the cultivation techniques used are not sufficient to obtain the desirable extracts. In this study, we compared the contents of the extract and the quantitative values of characteristic constituents obtained from wild-type and cultivated plants, and found a remarkable difference. Therefore, it is considered that these indicators play an important role in the establishment of better cultivation technology.

Identification of Antipneumococcal Molecules Effective Against Different Streptococcus pneumoniae Serotypes Using a Resazurin-Based High-Throughput Screen.

Streptococcus pneumoniae is a major human pathogen, causing around 1.6 million deaths worldwide each year. By optimizing a resazurin-based assay to detect S. pneumoniae growth in 384-well microplates, we developed a new high-throughput screening (HTS) system for the discovery of antipneumococcal molecules, which was unsuccessful using conventional absorbance measurements. Before applying our protocol to a large-scale screen, we validated the system through a pilot screen targeting about 7,800 bioactive molecules using three different S. pneumoniae serotypes. Primary screenings of a further 27,000 synthetic small molecules facilitated the identification of 3-acyl-2-phenylamino-1,4-dihydropquinolin-4-one (APDQ) derivatives that inhibited growth of S. pneumoniae with MIC90 values <1 µM (0.03-0.81 µM). Five selected APDQ derivatives were also active against Staphylococcus aureus but neither Klebsiella pneumoniae nor Pseudomonas aeruginosa, suggesting that APDQ may act specifically against Gram-positive bacteria. Our results both validated and demonstrated the utility of the resazurin-based HTS system for the identification of new antipneumococcal molecules. Moreover, the identified new antipneumococcal molecules in this study may have potential to be further developed as new antibiotics.

Two low complexity ultra-high throughput methods to identify diverse chemically bioactive molecules using Saccharomyces cerevisiae.

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue® (resazurin) or by direct optical measurement of cell growth. Both methods can be miniaturized to allow screening of large numbers of samples, and can be performed using S. cerevisiae in 384 and 1536-well format. The alamarBlue® approach achieves Z' values of >0.7 with signal to basal ratios of >6.5, and around 1.1 million low molecular weight compounds were screened, identifying approximately 25,000 primary hits. Dose response curves generated for a subset (1930) using both alamarBlue® and optical density methods showed significant overlap. In genome-wide haploinsufficiency profiling (HIP), 572 of these hits demonstrated a diverse mechanism of action, affecting >25% of all yeast strains.

[Simultaneous determination of paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in zhengtian pills by HPLC].

To simultaneously determine paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills. In the test, Insertil ODS-C18 column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the column temperature was 30 degrees C and the detection wavelength was 230 nm. According to the results of the test, paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol showed good linear relations between peak areas and sample sizes in 11.37-170.5, 2.188-32.82, 2.896-43.44, and 3.000-45.00 mg x L(-1) (r = 0.999 9, 0.999 9, 1.000 0, 1.000 0), respectively. The average recoveries (n = 6) were 102.3% (RSD 1.2%), 99.71% (RSD 1.9%), 101.2% (RSD 1.2%), and 99.40% (RSD 2.4%), respectively. The above four components were determined in five batches of samples by using the established method, with satisfactory results. The method was so simple, accurate and highly reproducible that it could be used for quality control of the four components in Zhengtian pills.

High-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry analysis of Radix Saposhnikoviae for metabolomic research.

In this study, metabolite profiling of Radix Saposhnikoviae from different geographical locations was performed using high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOFMS) and multivariate statistical analysis technique. Principle component analysis (PCA) of the data shows that these samples could be roughly separated into three groups: Guan Fangfeng, Kou Fangfeng and Chuan Fangfeng. The potential chemical markers were discovered through the loading plot of PCA. Based on accurate mass measurements and subsequent fragment ions of TOFMS after in-source collision induced dissociation, as well as matching of empirical molecular formulae with those of published components in the in-house chemical library, 10 potential markers, such as 4'-O-glucosyl-5-O-methylvisamminol, cimifugin, prim-O-glucosylcimifugin and 3'-O-angeloylhammaudol, were tentatively identified and partially verified by the available reference standards. The results of this study indicate that it is an effective and novel approach to identify traditional Chinese medicine (TCM) from different sources, and that performing quantity determination of corresponding marker compounds could optimize the quality control of TCM.

Isolated perfused lung extraction and HPLC-ESI-MS(n) analysis for predicting bioactive components of Saposhnikoviae Radix.

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\tandem mass spectrometry (HPLC-MS(n)) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC-ESI-MS(n). Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin (2) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin (1), 5-O-methylvisamminol (3) and sec-O-glucosylhamaudol (5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC-ESI-MS(n) for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable.

[Determination of prim-O-glucosylcimifugin and 5-O-methylvisammisoide in Saposhnikovia divaricata and HPLC fingerprint analysis].

OBJECTIVE: To develop HPLC methods for the determination of prim-O-glucosylcimifugin and 5-O-methylvisammisoide in Saposhnicovia divaricata and of HPLC fingerprint to compare the wild and culture varieties. METHOD: Conditions of determination: Shimadzu C18 column (4.6 mm x 150 mm, 5 microm), methanol-water (40:60) as mobile phase with the flow rate of 1 mL x min(-1). The detection wavelength was 254 nm. Conditions of HPLC fingerprint: MG II C18 column (4.6 mm x 250 mm, 5 microm), the mobile phase was acetonitrile-water with the flow rate of 1 mL x min(-1), using linear gradient elution, the column temperature was 30 degrees C. RESULT: The average recovery of prim-O-glucosylcimifugin was 99.6% (RSD 0.72%, n=6). The average recovery of 5-O-methylvisammisoide was 102.6% (RSD 0.88%, n=6). The contents of prim-o-glucosylcimifugin in wild and culture varieties were (4.96 +/- 2.59) and (3.61 +/- 1.82) mg x g(-1) respectively. The contents of 5-O-methylvisammisoide were (3.91 +/- 2.09) and (4.37 +/- 2.02) mg x g(-1) respectively. The compositions of S. divaricata were effective separated under the conditions of HPLC fingerprint. CONCLUSION: The HPLC determination method of prim-O-glucosylcimifugin and 5-O-methylvisammisoide is convenient and accurate. The HPLC fingerprint analysis method could be a basis for quality control and classification evaluate of S. divaricata.

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay.

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC(50) values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC(50) values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.

[Investigation on one-pot extraction of active ingredient group from Yupingfeng powder by HPLC].

The extraction of active ingredient group, i.e., prim-o-glucosylcimifugin, astragaloside, and 5-o-methylvisammiosode from Yupingfeng powder with one-pot method was studied in this work. A HPLC method was used to determine the content of each active constituent mentioned above in the extract. The influences of extraction temperature, time, volume percent of ethanol in water and its amount added on the content and yield of active ingredient group were investigated by orthogonal test. The experimental results showed that the optimized extraction conditions were as follows: 1g of Yupingfeng powder was one-pot extracted for 4 hours at 80 degrees C with 90% ethanol as solvent, and the yield and content of active ingredient group were 0.16%, 0.53% respectively. The active ingredient group in Yupingfeng powder could be effectively one-pot extracted under the conditions above.

[Studies on flavonoids from stems and leaves of Calophyllum inophyllum].

OBJECTIVE: To study the chemical constituents from the stems and leaves of Calophyllum inophyllum. METHOD: The compounds were isolated by column chromatography on silica gel, Sephadex LH-20 and preparative TLC. Their structures were elucidated by chemical methods and NMR, MS spectroscopic data. RESULT: Nine compounds were identified as 2-hydroxyxanthone (1), 4-hydroxyxanthone (2), 1, 5-dihydroxyxanthone (3), 1, 7-dihydroxyxanthone (4), 1, 3, 5-trihydroxy-2-methoxyxanthone (5), 6-deoxyjacareubin (6), amentoflavone (7), kaempferol-3-O-alpha-L-rhamnoside (8) and quercetin-3-O-alpha-L-rhamnoside (9). CONCLUSION: Compounds 8 and 9 were isolated from the genus Calophyllum and compounds 1, 2, 4-6 were isolated from this plant for the first time.

[Simultaneous analysis of six effective components in the anti-Alzheimer's disease effective component group of Xiao-Xu-Ming Decoction].

A method based on high performance liquid chromatography (HPLC) was developed for the quantitative determination of six components in an anti-Alzheimer medicine, Xiao-Xu-Ming Decoction, which is an effective prescription in treating stroke and the sequela of stroke by herbalist doctors for thousands years. The effective component group (ECG) was made according to the results of high-throughput screening, and the curative effect of ECG was validated on aging rats. In this method an ODS column was used. The mobile phase consisted of water-formic acid-ethylenediamine (A; 100: 0.1: 0.1, v/v) and methanol-formic acid (B; 100 : 0.05, v/v), eluted with gradient (0 - 5 min, 20% B; 5 - 100 min, 20% B - 40% B; 100 - 140 min, 40% B - 70% B). The flow rate was 1 mL/min. The detection wavelength was set at 240 nm. Under the above separation conditions, six components belonged to two different categories, indicans and alkaloids, were determined simultaneously. The relationships between the concentrations and the peak areas of these six components were all linear. The recoveries of the six components were 99.1% for paeoniflorin, 99.6% for prim-O-glucosylcimifugin, 98.4% for baicalin, 99.9% for 4'-O-beta-D-glucosyl-5-O-methylvisamminol, 99.6% for fangchinoline, and 102.0% for tetrandrine. The relative standard deviations (RSD) were 1.3%, 1.4%, 0.4%, 0.8%, 0.2%, and 1.4%, respectively. This method is simple and reproducible and it can be used for the quality control of the effective component group of Xiao-Xu-Ming Decoction.

[Determination of four components in root of Saposhnikovia divaricata by HPLC gradient elution].

OBJECTIVE: To develop an HPLC method for determination of components in root of Saposhnikovia divaricata. METHOD: The separation of four components was achieved on a reversed-phase Alltima C18 column with MeOH-H2O gradient elution at a flow rate of 1.0 mL x min(-1). RESULT: Six samples were determined and there is no distinct difference between the contents determined by the new and old methods. CONCLUSION: The new method is more simple and convenient than the old method.

Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay.

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

Isolation and quantitative analysis of phenolic antioxidants, free sugars, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement.

An aqueous decoction of mango (Mangifera indica L.) stem bark has been developed in Cuba on an industrial scale to be used as a nutritional supplement, cosmetic, and phytomedicine. Previously we reported its antioxidant activity, and we concluded that the product could be useful to prevent the production of reactive oxygen species and oxidative tissue damage in vivo. A phytochemical investigation of mango stem bark extract has led to the isolation of seven phenolic constituents: gallic acid, 3,4-dihydroxy benzoic acid, gallic acid methyl ester, gallic acid propyl ester, mangiferin, (+)-catechin, (-)-epicatechin, and benzoic acid and benzoic acid propyl ester. All structures were elucidated by ES-MS and NMR spectroscopic methods. Quantitative analysis of the compounds has been performed by HPLC, and mangiferin was found to be the predominant component. Total polyphenols were assayed also by the Folin-Ciocalteu method. The free sugars and polyols content was also determined by GC-MS.

[Polyphenol constituents from Salacia species: quantitative analysis of mangiferin with alpha-glucosidase and aldose reductase inhibitory activities].

Mangiferin, three catechins, and two catechin dimers were isolated from the roots of Salacia reticulata (SRE), and examined their inhibitory activities against several carbohydrate metabolize enzymes (sucrase, maltase, isomaltase, alpha-amylase, and aldose reductase). Among them, mangiferin was found to inhibit sucrase, isomaltase, and aldose reductase from rat with IC50 values of 87, 216 and 1.4 micrograms/ml, respectively. The inhibitory activities of mangiferin are competitive for sucrase and isomaltase with inhibitor constant (Ki) 55 micrograms/ml and 70 micrograms/ml, respectively. In order to determine the mangiferin contents in the water extracts from the roots of S. reticulata, a quantitative analytical method by means of HPLC was developed and the mangiferin contents in SRE were determined to be in the range of 0.9-2.3% by the application of this method. A high linear correlation (r = 0.934) was observed between the mangiferin contents and the sucrase inhibitory activity. In addition, this analytical procedure of mangiferin was found to be applicable for other Salacia species (S. oblonga, S. chinensis, and S. prinoides). Thus, the quantitative HPLC analysis of mangiferin was supposed to be suitable for the quality control of Salacia species and its products.

Xanthones and triterpenoids from the bark of Garcinia vilersiana.

The hexane extract of the bark of Garcinia vilersiana from Vietnam contained four triterpenoids (olean-12-ene-3beta,11alpha-diol, lupeol, beta-amyrin and oleanolic acid), and six xanthones (globuxanthone, subelliptenone H, subelliptenone B, 12b-hydroxy-des-D-garcigerrin A, 1-O-methylglobuxanthone and symphoxanthone). The structure of 1-O-methylglobuxanthone, the only novel compound, was determined using 1D and 2D NMR techniques and by correlation with globuxanthone.

[Effect of processing on chemical composition and medical activity of rhizoma Anemarrhenae].

A comparative study was conducted on the chemical composition and medical activity of different kinds of processed drug of Rhizoma Anemarrhenae. It was found that the content of effective components of zhimu was affected obviously by processing: the rhizome should not be barked when used as drug. It was also observed that different kind of processed drug should be used in line with different particular circumstance.

Loading and localization of Fluo-3 and Fluo-3/AM calcium indicators in sinapis alba root tissue.

Stimulus-induced changes in free cytosolic Ca2+ in different types of plant cells have been monitored with the aid of fluorescent calcium indicator dyes. However, there is no simple and convenient method for introducing these dyes into the plant cell cytoplasm. This paper reports tests of different procedures for loading either free fluorescent dyes or their acetoxymethyl esters (Fluo-3 and Fluo-3/AM, respectively) into Sinapis alba root tissue. Loading of Fluo-3 was pH and temperature dependent. Moreover, in the presence of beta-escin (saponin) in the loading medium very high fluorescent signals in root tissues were observed. The highest signals were recorded when tissue was loaded in a medium containing 0.1% beta-escin, at pH 5.0 and 30 degrees C. Only very weak fluorescence signals were found in mustard roots loaded with Fluo-3/AM. Acidity and temperature of the medium had no significant effect on the process. However, addition of eserine, a cholinesterase inhibitor led to a dramatic increase in fluorescence in the root cells. On the basis of these observations rapid and efficient methods of loading both Fluo-3 and Fluo-3/AM into mustard root tissues are proposed.

[Determination of mangiferin in qingqiliangying injection by reversed-phase HPLC].

A reversed-phase HPLC method has been established for the determination of mangiferin in Qingqiliangying Injection. The mangiferin was separated on a Nova-pak C18 column (150 mm x 3.9 mm) and detected at 258 nm, using methanol-tetrahydrofuran-0.85% H3PO4 aqueous solution (500:80:15) as the mobile phase. The average recovery of mangiferin was 100.9%. The method is simple, rapid, and well reproducible, and thus very reliable for the quality control of Qingqiliangying Injection.

Determination of gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in the traditional Chinese medicinal preparation sann-joong-kuey-jian-tang by high-performance liquid chromatography.

High-performance liquid chromatography was employed to determine the contents of several marker substances such as gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in Sann-Joong-Kuey-Jian-Tang. The separation was performed on a Cosmosil 5C18-AR column by gradient elution with 0.03% (v/v) phosphoric acid-acetonitrile (0 min, 90:10; 10 min, 87:13; 17-27 min, 77:23; 40 min, 62:38; 50 min, 55:45) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. n-Propylparaben was used as the internal standard and seven regression equations revealed linear relationships between the peak-area ratios (marker substances/internal standard) and concentrations. The repeatability and reproducibility (relative standard deviation) of the method were in the ranges 0.02-1.78% and 1.44-4.95%, respectively.