Rapid screening for natural lipase inhibitors from Alisma orientale combining high-performance thin-layer chromatography-bioautography with mass spectrometry.

Lipase inhibitors are an attractive class of hypolipidemic compounds, which inhibit the activity of human pancreatic lipase, thereby preventing the absorption of triglycerides in vivo. As a library of promising lead compounds for drug development, traditional Chinese medicine (TCM) has gained growing attention in quick discovery and identification of enzyme inhibitors of natural-origin. The purpose of this work was to discover unknown lipase inhibitors from Alisma orientale by the activity oriented analysis method thin-layer chromatography-bioautography, then use electrospray ionization mass spectrometry technology via the elution based TLC-MS interface to identify their structures. As a result, eleven natural lipase inhibitors from Alisma orientale extracts were identified based on molecular mass and fragment ions obtained by HPTLC-MS, and further confirmed by a series of complementary means including UV spectra, 1H NMR characteristic proton signals and polarity of compounds, eleven lipase inhibitors were tentatively assigned as triterpenoids: alisol B (m/z 495.50 [M + Na]+), alisol B 23-acetate (m/z 537.58 [M + Na]+), 11-deoxy-alisol B (m/z 479.50 [M + Na]+), 11-deoxy-alisol B 23-acetate (m/z 521.50 [M + Na]+), alisol A/epialisol A (m/z 513.50 [M + Na]+), 16-oxo-11-deoxy-alisol A (m/z 511.50 [M + Na]+), 16-oxo-alisol A (527.50 [M + Na] +), alisol C (m/z 509.58 [M + Na]+), alisol C 23-acetate (m/z 551.50 [M + Na]+), alisol M 23-acetate (m/z 567.50 [M + Na]+), and alismanol Q/neoalisol (m/z 493.42 [M + Na]+). The integrated approach is an efficient method for rapid screening lipase inhibitors from complex plant extracts and provides a reasonable and favorable basis for the identification and separation of other enzymatic system and other important compounds with therapeutic values.

An integrated approach to uncover quality marker underlying the effects of Alisma orientale on lipid metabolism, using chemical analysis and network pharmacology.

BACKGROUND: Quality control of traditional Chinese medicines is currently a great concern, due to the correlation between the quality control indicators and clinic effect is often questionable. According to the "multi-components and multi-targets" property of TCMs, a new special quality and bioactivity evaluation system is urgently needed. PURPOSE: Present study adopted an integrated approach to provide new insights relating to uncover quality marker underlying the effects of Alisma orientale (AO) on lipid metabolism. METHODS: In this paper, guided by the concept of the quality marker (Q-marker), an integrated strategies "effect-compound-target-fingerprint" was established to discovery and screen the potential quality marker of AO based on network pharmacology and chemical analysis. Firstly, a bioactivity evaluation was performed to screen the main active fractions. Then the chemical compositions were rapidly identified by chemical analysis. Next, networks were constructed to illuminate the interactions between these component and their targets for lipid metabolism, and the potential Q-marker of AO was initially screened. Finally, the activity of the Q-markers was validated in vitro. RESULTS: 50% ethanol extract fraction was found to have the strongest lipid-lowering activity. Then, the network pharmacology was used to clarify the unique relationship between the Q-markers and their integral pharmacological action. CONCLUSION: Combined with the results obtained, five active ingredients in the 50% ethanol extract fraction were given special considerations to be representative Q-markers: Alisol A, Alisol B, Alisol A 23-acetate, Alisol B 23-acetate and Alisol A 24-acetate, respectively. The chromatographic fingerprints based Q-marker was establishment. The integrated Q-marker screen may offer an alternative quality assessment of herbal medicines.

Description of analytical method and clinical utility of measuring serum 7-alpha-hydroxy-4-cholesten-3-one (7aC4) by mass spectrometry.

BACKGROUND: Imbalance of bile acids (BA) homeostasis in the gastrointestinal tract can lead to chronic diarrhea or constipation when BA in the colon are in excess or low, respectively. Since both disturbances of bowel function can result from other etiologies, identifying BA imbalance is important to tailor treatment strategies. Serum concentrations of 7-alpha-hydroxy-4-cholesten-3-one (7aC4), a precursor in bile acid synthesis, reflect BA homeostasis. Here we describe a method to accurately measure serum 7aC4 and evaluate the clinical utility in patients with diarrhea or constipation phenotypes. METHODS: Serum 7aC4 is measured after acetonitrile protein precipitation using C18 liquid chromatography, tandem mass spectrometry, and deuterium-labeled 7aC4 internal standard. Assay performance including linearity, precision, and accuracy was assessed using waste serum samples. The reference interval was established in healthy individuals without BA-altering conditions or medications. Clinical performance was assessed in patients with irritable bowel syndrome. RESULTS: The method precisely and accurately measured 7aC4 in human serum from 1.4-338ng/mL with no ion suppression or interference from related 7-keto-cholesterol. Central 95th percentile reference interval was 2.5-63.2ng/mL. Lower serum 7aC4 was found in patients with constipation with sensitivity/specificity of 79%/55% compared to healthy controls. Higher 7aC4 was found in patients with bile acid diarrhea (BAD) compared to those without BAD with sensitivity/specificity of 82%/53%. CONCLUSIONS: We have developed a sensitive and precise assay for measuring the concentration of 7aC4 in serum. The assay can be used to screen for diarrhea caused by bile acid malabsorption.

The difference between blood-associated and water-associated herbs of Danggui-Shaoyao San in theory of TCM, based on serum pharmacochemistry.

Danggui-Shaoyao San (DSS) is a famous Chinese formula for activating blood circulation and promoting urination. This study was to investigate the difference of material basis between a blood-associated herbs group and a water-associated herbs group. According to the theory of traditional Chinese medicine, the formula can be divided into a blood-associated herbs group (Angelica sinensis, Paeonia lactiflora and Ligusticum chuanxiong) and a water-associated herbs group (Atractylodes macrocephala, Alisma orientale and Poria cocos). The HPLC fingerprint of the formula was established for quality control. Serum samples from rats, orally administrated DSS, and the decomposed recipes of DSS, were analyzed by HPLC-DAD and the transitional blood components of DSS were identified. Twenty-one common peaks were identified in the fingerprint of DSS. Contents of paeoniflorin, albiflorin, ferulic acid and alisol B 23-acetate in co-decoction were significantly higher than those in individual decoction. Eleven peaks belonged to the blood-associated herbs group (four metabolites and seven prototype components; paeoniflorin and ferulic acid appeared in prototype components), whereas six peaks belonged to the water-associated herbs group (three metabolites and three prototype components). It was concluded that the serum pharmacochemistry is a meaningful approach for clarifying the difference between blood-associated and water-associated herbs in chemical composition.

[Optimization of flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma by response surface methodology].

Response surface methodology was used to optimize and obtain the optimal flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma. With the extraction rate of alisol B 23-acetate as an indicator, single-factor test was used to investigate the effect of ethanol volume fraction, liquid-solid ratio, extraction times and extracting time on the extraction rate of alisol B 23-acetate.The results were combined with Box-Benhnken design and response surface analysis to optimize the technology parameters for extraction process of Alismatis Rhizoma and obtain the optimal flash-type extraction technology under the following conditions： ethanol volume fraction 80%, liquid-solid ratio 12∶1, extraction 4 times, 114 s/time. Flash-type extraction technology of alisol B 23-acetate by response surface methodology is stable, time-saving, efficient, and with the advantages of room temperature extraction and no component damage, so it can be used for massive production.

A simultaneous determination of principal compounds in tokishakuyakusan by high-performance liquid chromatography with diode array detector.

We developed a simultaneous analysis method using high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD) for six principal compounds (atractylenolide III, alisol A, alisol B, paeoniflorin, ferulic acid and (Z)-ligustilide) in a traditional Japanese (Kampo) medicine, tokishakuyakusan (TSS). The HPLC separation was conducted on a reversed-phase TSK-gel ODS-80TS column (4.6 i.d. × 250 mm, 5 µm) at 40°C with a 0.1% phosphoric acid-acetonitrile gradient system. The DAD detection wavelength was set at 205, 232 and 330 nm. Calibration curves for the compounds showed linear regressions with correlation coefficients of >0.999. The intra- and inter-day precision (i.e., the relative standard deviation) were in the range of 0.50-1.55 and 0.70-1.80%, respectively. The average recovery yields of the compounds ranged from 98.3 to 103%. The present results will contribute to shorter analysis times with less organic solvent compared with the individual analysis of each compound for the evaluation of TSS. The application of the established method to TSS will also provide helpful information for the further pharmacological and clinical studies.

The single-probe: a miniaturized multifunctional device for single cell mass spectrometry analysis.

We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 µm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.

A sensitive spectrofluorometric method for determination of ergosta-4,6,8(14),22-tetraen-3-one in rat plasma, feces, and urine for application to pharmacokinetic studies using cerium(III) as a probe.

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) isolated from Polyporus umbellatus possesses a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic, and immunosuppressive effect. The interaction of cerium ions (Ce(3+)) with ergone was studied by fluorescence and absorption spectroscopy. Spectra data revealed that Ce(3+) ions exhibited emission maxima around 350 nm when the excitation wavelength was fixed at 255 or 290 nm, and the fluorescence of Ce(3+) ions was quenched by the addition of ergone, indicating that a Ce(3+)-ergone complex was formed. According to the modified Benesi-Hildebrand equation, the binding constant of interaction of Ce(3+) ions with ergone was obtained at room temperature. Based on this, a sensitive spectrofluorometric method using Ce(3+) ions as a probe was applied for the identification and quantification of ergone in rat plasma, feces, and urine. The linear ranges of the calibration curves were 1.31 to 4.50 µM for plasma, 1.12-9.87 µM for feces, and 1.28-3.42 µM for urine, and the ergone recoveries were found to be 97.1 ± 0.9%, 98.2 ± 0.7% and 96.5 ± 1.4% for plasma, feces, and urine, respectively. The intraday and inter-day relative standard deviations were less than 9.7%. The proposed spectrofluorometric method is simple and rapid for the quantitative determination of ergone in rat plasma, feces, and urine, and it is affordable for most laboratories because it has few requirements and uses low cost, easy to operate equipment.

Study on the hypoglycemic activities and metabolism of alcohol extract of Alismatis Rhizoma.

The Alisma rhizoma is widely used in the therapy of diabetes in traditional folk medicine of China. Compositional analysis of the alcohol extract of Alismatis Rhizoma (AEA) revealed that the eight compounds gotten from AEA are all belonging to protostane-type triterpenes. The AEA and compounds were incubated with 3T3-L1 preadipocytes, glucose level in the 3T3-L1 adipocytes culture medium and lipid content in 3T3-L1 adipocytes were measured, and analysis of alpha-glucosidase inhibition of AEA and compounds. At the same time, the uptake of AEA by 3T3-L1 adipocytes and the metabolism of AEA in SD rats were analyzed by HPLC-ESI/MS. As result, AEA increased glucose uptake in 3T3-L1 adipocyte model, not increase adipogenesis; AEA inhibited alpha-glucosidase activity; alisol A-24-aceate (8) was absorbed by 3T3-L1 adipocytes; and two compounds were detected in blood and three were detected in urine in SD rats. So AEA had protostane-type triterpenes, these type compounds in AEA may have hypoglycemic activity via inhibition of alpha-glucosidase activity and promotion of glucose uptake. In contrast to the anti-diabetic drug thiazolidinediones, they did not induce adipogenesis, avoiding the displeased effects of rosiglitazone.

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d7)-standards of 7-DHC and DHCEO were synthesized from d7-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d7-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d7-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3ß-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3ß,5α,6ß-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.

[Chemical constituents from Sarcandra glabra].

OBJECTIVE: To study the chemical constituents of the plant of Sarcandra glabra and provide reference for the study of the bioactive substances. METHOD: The compounds were isolated from the EtOH extract by various chromatographic methods and their structures were elucidated by their physico-chemical properties and the analysis of their spectral data. RESULT: Nine compounds were isolated and identified as isoscopletin (1), syringaresinol monoside (2), styraxjaponoside B (3), 5-O-caffeoylshikimic acid (4), shizukanolide E (5), isoastilbin (6), neoisoastilbin (7), astilbin (8), neoastilbin (9). CONCLUSION: Compounds 1-7 were isolated from S. glabra for the first time.

[Simultaneous determination of four alisols in Rhizoma Alismatis by RP-HPLC].

OBJECTIVE: To develop an HPLC method for the determination of alisol A, alisol F, alisol A 24-acetate and alisol B 23-acetate in the rhizome of Alisma orientalis. METHOD: The separation was performed on a Shim-Pack CLC-ODS a C18 column (6 mm x 150 mm, 5 microm) eluted with the mobile phases of acetonitrile (A)-water (B) in gradient elution. The absorbance was monitored at 210 nm. Orthogonal test was adopted to optimize the extraction conditions of the four alisols. RESULT: The correlation coefficients of the four alisols were higher than 0.999 and the average recoveries were 99.23%, 96.67%, 97.30% and 99.61%, respectively. All the RSDs were less than 3%. CONCLUSION: The validation data demonstrated that the method was accurate and repeatable, and can be used to measure the four alisols in Rhizoma Alismatis.

Increased atherosclerosis and vascular inflammation in APP transgenic mice with apolipoprotein E deficiency.

OBJECTIVE: Atherosclerosis is associated with Alzheimer's disease (AD) in humans, but the nature of this link is still elusive. Aim of this study was to investigate aortic atherosclerosis development in a mouse model with central nervous system (CNS) restricted beta-amyloid precursor protein (APP) overexpression. METHODS AND RESULTS: APP23 mice, overexpressing the Swedish mutated human APP selectively in the brain, were crossed with mice lacking apolipoprotein E (ApoE KO). Nine weeks old mice were fed a western type diet for eight weeks, then atherosclerotic lesions, aortic wall and cortical tissues gene expression and beta-amyloid (Abeta) deposition were evaluated. Compared with ApoE KO, APP23/ApoE KO mice developed larger aortic atherosclerotic lesions and showed significantly increased expression of MCP-1, IL-6, ICAM-1 and MTPase 6, a marker of oxidative stress in the vascular wall. Of note brain limited APP synthesis was associated with an increased microglia and brain endothelial cells activation, in spite of the absence of beta-amyloid deposits in the brain or alteration in the levels of oxidized metabolites of cholesterol such as 4-cholesten-3-one. CONCLUSION: Our study suggests that the vascular pro-inflammatory effects of CNS-localised APP overexpression lead to atherogenesis before parenchymal Abeta deposition and neuronal dysfunction.

[Studies on chemical components of Lobelia chinensis].

OBJECTIVE: To study on the chemical constituents of Lobelia chinensis. METHOD: The coloumn chromatographic techniques were applied to isolate constituents, and their structures were elucidated by means of spectral data analysis. RESULT: Sixteen compounds were isolated and identified as daucosterol (1), diosmetin (2), apigenin (3), chrysoeriol (4), loteolin (5), hesperidin (6), loteolin-7-O-beta-D-glucoside (7), apigenin-7-O-beta-D-glucoside (8), linarin (9), diosmin(10), 5,7-dimethoxy-8- hydroxycoumarin (11), palmitinic acid (12), lacceroic acid (13), stearic acid (14), beta-sitosterol (15), daucosterol (16). CONCLUSION: All of these compouds were obtained from L. chinensis for the first time.

[Variation in yield and quality of Alisma orientalis grown under different ecological climatic regions].

UNLABELLED: To compare yield, alisol content of Alisma orientalis planted at different ecological climatic regions, and explore further the impact of environmental factors on the yield and quality. METHOD: Different local varieties were planted at varing ecological climatic conditions. Diameter, yield was measured after harvest, the contents of 23-acetyl alisol B and 24-acetyl alisol A were quantitatively analyzed by HPLC. RESULT: The result revealed that ecological condition had significant impacts on yield and alisol content. Yield of MeiShan was the highest which was up to 1 200.72 kg x hm(-2). The contents of 23-acetyl alisol B and 24-acetyl alisol A of A. orientalis cultivated in Dujiangyan were significantly higher than those of other regions, the values were up to 4.222, 2.727 g x kg(-1), respectively. 23-acetyl alisol B content was positively correlated with 24-acetyl alisol A content (P < 0.01). The diameter was positively correlated with yield (P < 0.01). CONCLUSION: Considering yield and medicinal ingredients, Dujiangyan may be the most suitable region to plant A. orientalis.

Faecal steroid excretion in humans is affected by calcium supplementation and shows gender-specific differences.

BACKGROUND: Previous human studies on the effect of dietary calcium supplementation on faecal excretion of bile acids (BA) and faecal water concentrations of animal neutral sterols (NSt, cholesterol and its metabolites) lack detailed information about single BA and NSt. AIM OF THE STUDY: We investigated whether single BA and NSt in faeces and especially in faecal water are affected by calcium supplementation and whether this affects genotoxicity of faecal water. In addition, we differentiated between men and women with regard to the concentrations of BA and NSt in faecal water. METHODS: Thirty-one healthy volunteers consumed a calcium supplemented bread (1.0 g/day) and a placebo bread, respectively, for 4 weeks in a double-blind, randomised cross-over trial. Faeces were collected quantitatively for 5 days in the last week of each period. NSt and BA were analysed by GC-MS. RESULTS: Due to calcium supplementation faecal concentrations of lithocholic acid (LCA, 14%, P = 0.008), deoxycholic acid (DCA, 19%, P < 0.001) and 12 keto-deoxycholic acid (12 keto DCA, 29%, P = 0.049) significantly increased whereas BA concentrations in faecal water were only marginally affected. In contrast, concentrations of cholesterol (30%, P = 0.020) and its metabolites coprostanol (43%, P = 0.004), coprostanone (36%, P = 0.003), cholestanol (44%, P = 0.001) and cholestenone (32%, P = 0.038) in faecal water significantly decreased. Total NSt concentration in faecal water was found to be significantly higher in women compared to men (P = 0.018). The genotoxicity of faecal water was neither affected by calcium supplementation nor were there gender-specific differences. CONCLUSIONS: Dietary calcium supplementation diversely affects BA and NSt in faeces and in faecal water but does not influence the genotoxicity of faecal water in healthy adults.

[Effects of S-3307 on the yield and main ingredients of Alisma plantago-aquatica].

UNLABELLED: To study the effect of S-3307 on the yield and main ingredients of Alisma plantago-aquatica. METHOD: The contents of 24-acetyl alisol A and the 23-acetyl alisol B in tuber were determined by HPLC. RESULTS: The contents of 24-acetyl alisol A and the 23-acetyl alisol B as well as yield were significantly increased in all groups applied with different concentrations of S-3307 comparing with control group. The optimal concentration of S-3307 was 80 mg x kg(-1). The residues of S-3307 was detected under 0.316 8 mg x kg(-1) (detecting limit). CONCLUSION: The optimal concentration of S-3307 is 80 mg x kg(-1), it reached the best result when applied 36 d after seedling.

[Establishment of chromatographic fingerprint and quality assessment on Sichuan native medicinal plant Alisma plantago-aquatica].

OBJECTIVE: To establish chromatographic fingerprint of Sichuan native medicinal plant Alisma plantago-aquatica by RP-HPLC for the quality control. METHOD: The gradient elution mode was applied in chromatographic separation, and data were analyzed by "Computer Aided Similarity Evaluation" software to compare the quality of A. plantago-aquatica samples from different habitats. RESULT: The HPLC chromatographic fingerprinting of A. plantago-aquatica with 26 characteristic peaks was established from 17 lots of A. plantago-aquatica samples, peak 16 and 22 were identified as 24-acetyl alisol A and 23-acetyl alisol B, respectively. CONCLUSION: The chromatographic fingerprinting of A. plantago-aquatica with high specificity can be used to control its quality and assure lot to lot consistency. The RP-HPLC fingerprint method is repeatable, feasible in analysis of A. plantago-aquatica.

Electron ionization mass spectral fragmentation of derivatized 4,5- and 5,6-epoxysterols.

The electron ionization (EI) mass spectral fragmentation of derivatized 4,5- and 5,6-epoxysterols was investigated. Interesting fragmentation processes involving a transannular cleavage of the epoxide ring after transfer of the trimethylsilyl group are significant in the case of 4,5-epoxysterol trimethylsilyl ethers (affording abundant fragment ions at m/z 403 and 404). Different pathways, which have been substantiated by deuterium labelling, are proposed in order to explain the formation of these ions. In contrast, this transfer is not significant in the case of 5,6-epoxysterol trimethylsilyl ethers. The EI mass spectra of these latter compounds appear to be very complex and to differ slightly according to the stereochemistry of the epoxy group. Acetate and trifluoroacetate derivatives of 4,5-epoxysterols display interesting EI mass spectra dominated by a fragment ion at m/z 332 resulting from cleavage of the steroid ring A.

[Studies on the marker compounds for standardization of traditional Chinese medicine "polyporus sclerotium ([symbol: see text])"].

Natural products have been used for healthcare and pharmacotherapy. Because difficulties in quality control affect their production, processing, and marketing, it is necessary to establish adequate marker compounds for their effective application. Ergosta-4,6,8(14),22-tetraen-3-one(I) was studied in the screening of the marker compounds for the standardization of Polyporus Sclerotium ([symbol: see text]), which has the advantage of easy qualitative and quantitative analysis because of its fluorescence. Its applicability in the standardization of Polyporus Sclerotium is discussed based on comparative studies of 30 crude samples of Polyporus Sclerotium and some other fungi herbs using TLC and HPLC analysis with I it as the marker compound, as well as its chemical synthesis.