PhytochemicalinvestigationofleavesofDendrobiumofficinale / 中草药

Objective: To investigate the chemical constituents of leaves of Dendrobiumofficinale. Methods: Compounds were isolated from the leaves of D.officinale by column chromatography over Sephadex LH-20, MCI GEL CHP-20P, and ODS as well as by preparative HPLC. Their structures were identified by the analysis of their physical and chemical properties and the spectra data of NMR and MS. Results: Twenty-four compounds were isolated from the leaves of the plant, namely 3,4-dihydroxy-5,4'-dimethoxy bibenzyl (1), moscatilin (2), 4,4'-dihydroxy-3,5-dimethoxybibenzyl (3), densiflorol A (4), (S)-3,4,α-trihydroxy-5,4'-dimethoxybibenzyl (5), gigantol (6), dendrocandin U (7), dendrocandin B (8), loliolide (9), (6R,9S)-dihydroxy-megastigma-4,7-dien-3-one-9-O-β-D-glucopyranoside (10), (6R,9S)-9-hydroxy-megastigma-4,7-dien-3-one-9-O-β-D-glucopyranoside (11), (+)-syringaresinol (12), rutin (13), 2-benzothiazolol (14), p-hydroxyacetophenone (15), p-hydroxyl-benzoic acid (16), protocatechuic acid (17), catechol (18), ethyl p-hydroxyhydrocinnamate (19), 1-glycerol linolenate (20), 2-butoxyethyl linolenate (21), palmitic acid (22), octadecadienoic acid-2,3-dihydroxypropyl ester (23), and urticifolene (24). Conclusion: Itis the first report of the occurrence of compounds 10, 11, 14-23 in Orchidaceae family. Compounds 1, 2, 4, and 6are found in D. officinale for the first time.

NMR-based study of chemical constituents of Massa Medicata Fermentata / 中草药

Objective: To study the chemical constituents from Medicated Leaven (namely Massa Medicata Fermentata in Chinese). Methods: Compounds were isolated and purified from chloroform extract of Medicated Leaven by column chromatograph of silica gel, ODS, Sephadex LH-20, HPLC, and their structure were elucidated on the basis of spectral data and physicochemical property. Results: Eleven chemical compounds were identified as 5-pentacosyl resorcinol (1), 5-heptadecyl resorcinol (2), 5-heneicosyl resorcinol (3), 5-tricosyl resorcinol (4), (9S,10E,12Z)-9-hydroxy-10,12-octadeca-dienoic acid (5), trans-4-hydroxy-2-nonenoic acid (6), 9,10,11-trihydroxy-11(E)-octadecenoic acid (7), (9S,12R,13S)-9,12,13-trihydroxy-10(E)-octadecenoic acid (8), (9S,12S,13S)- 9,12,13-trihydroxy-10(E)-octadecenoic acid (9), (9S,12S,13S)-9,12,13-trihydroxy-10(E)-octadecenoic acid methyl ester (10), and 9,12(Z)-octadecadienoic acid methyl ester (11). Conclusion: All above compounds are all isolated from Massa Medicata Fermentata for the first time, and the chloroform extract of Massa Medicata Fermentata and compound 8 showed inhibitory effect against TNF-α generation of RAW 264.7 induced by LPS in a dose-dependent manner.

Chemical constituents from stems of Uncaria lancifolia / 中草药

Objective To study the chemical constituents from the stems of Uncaria lancifolia. Methods The isolation and purification were carried out by silica gel column chromatography, RP18, Sephadex LH-20, and preparative HPLC. The structures of the isolated compounds were elucidated by physical and chemical properties, and spectroscopic methods. Results Eighteen compounds were isolated and identified from 95% ethanol extract from the stems of U. lancifolia and characterized as uncarine A (1), uncarine E (2), isomitraphylline (3), tetrahydroalstonine (4), strictosidine (5), cadambine (6), glabratine (7), strictosamide (8), (13R)-hydroxy- octodeca-(9Z,11E,15Z)-trien-oic acid (9), (6S,9R)-roseoside (10), periplanetin (11), integracin A (12), integracin B (13), 6β,19α- dihydroxyurs-3-oxours-12-en-28-oic aicd (14), ursolic acid (15), oleanic acid (16), β-sitosterol (17), and β-daucosterol (18). Conclusion This is the first report for the chemical constituents from U. lancifolia. All compounds are obtained from this plant for the first time, and compounds 9-13 are isolated from Uncaria genus for the first time. Compounds 1-8 are monoterpene indole alkaloids, which are characteristic constituents in Uncaria genus.

Comparative Study on Transformation of Tecomin by Using Liver Microsomes and Liver S9 QIAO Shan-shan, / 中国中医药信息杂志

Objective To compare the difference of transformation profile and transformation rate of tecomin by using two in vitro liver metabolism models. Methods Liver microsomes and liver S9 fraction models were employed to transform tecomin. HPLC was used to determine the contents of tecomin and its metabolites at the detecting wavelength of 254 nm. The gradient elution (0–6 min, 5％–40％ A; 6–9 min, 40％–50％ A; 9–11 min, 50％–5％ A) was carried out by using mobile phase of acetonitrile (A) - 1％ acetic acid (B) at a flow rate of 1 mL/min. Results Both models could transform tecomin into veratric acid; however, the metabolites obtained with liver S9 were more than those obtained with liver microsomes, and the transformation rate of the former was higher than that of the latter. Conclusion The liver S9 fraction can more efficiently transform esters than liver microsomes.

Evaluation for impacts of gut wall metabolism of multiple components in Chuanxiong Rhizoma during intestinal absorption with S9 incubation / 中国中药杂志

The information of drug deposition in the intestine is required in the study for the drug absorption in biopharmaceutics classification system (BCS). To illustrate the impacts of gut wall metabolism on the absorption, metabolism of multiple components in Chuanxiong Rhizoma in gut wall was tested by rat S9 incubation in vitro. The chemical fingerprint technology was used in this study to simultaneously detect multiple components in Chuanxiong, and peak areas before and after S9 incubation were compared. The results showed that senkyunolide I and several constituents were metabolized by gut wall, and one new metabolite was founded. However, ferulic acid and other compounds remained unchanged after incubation. Therefore, the subsequent intestinal permeability of multiple components in Chuanxiong that were not metabolized in the intestine was suggested to be detected directly by in situ single-pass intestinal perfusion (SPIP).Nonetheless, the intestinal permeability of the constituents that were metabolized in the intestine shall be explored by appropriate approaches.

Chemical constituents from Spiraea pubescens / 中草药

Objective: To investigate the chemical constituents from the ethanol extract of Spiraea pubescens. Methods: The compounds were isolated and purified by chromatography on silica gel, ODS, and preparative HPLC. Their structures were elucidated on the basis of chemical and spectroscopic methods, including MS, 1D, and 2D NMR spectral techniques. Results : Fourteen compounds were isolated and identified as β-sitosterol (1), tricosyl alcohol (2), stigmast-4-en-3-one (3), pentacosyl alcohol (4), stigmastanol (5), (+)-cyclo-olivil (6), (+)-africannal (7), (+)-lyoniresinol (8), 5-methoxy-(+)-isolariciresinol (9), (+)-isolariciresinol (10), (7R,8R)-4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan (11), (6S,9R)-6-hydroxy-3-one-α-ionol-9-O-β-D-glucopyranoside (12), (+)-lyoniresinol 9-O-β-D-xylopyranoside (13), and (-)-lyoniresinol 9-O-β-D-xylopyranoside (14). Conclusion: Compounds 2-5 and 7-14 are isolated from the plants of Spiraea L. for the first time, and compounds 1 and 6 are obtained from this plant for the first time.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R 2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Using spiral coating technique to evaluate the activity of rat liver S9 prepared by combination inducing method / 中国比较医学杂志

Objective To reduce experimental costs and improve the utilization of S9, we use spiral coating technique to evaluate the activity of rat liver S9 prepared by combination inducing method as well as to establish cryopreservation method.Methods Using spiral coating technique and Ames test to evaluate the activity of self-made rat liver S9 and commercially available S9 separately.We use glycerinum as protective agent to establish cryogenic storage method, so that S9 can be in liquid form stored at -20 °C in the refrigerator.Results In the Ames assay as well as using spiral coating technique, the number of revertant colonies had dose-response relationship among the dose of S9.When conditions were the same, the number of revertant colonies in positive control was at the approximate level in presense of self-made rat liver S9 and commercially available S9 respectively.When S9 ( concentration of 38%) was added to the amount at 1.48 ~6.62 μL /μL broth dose of bacteria, it can significantly induced Salmonella typhimurium histidine strains TA100, reverse mutation rates were three times more than the control group.Conclusions Spiral coating technique can successfully evaluate the activity of rat liver S9.The inducing method of combination of PB and BF can take the place of the unducing method of PCBs in the preparation of Liver homogenate S9.

Study on chemical constituents from whole herbs of Xanthium mongolicum / 中草药

Objective: To study the chemical constituents in the whole herbs of Xanthium mongolicum. Methods: The chemical constituents were isolated and purified by chromatography on silica gel column and HPLC, and their structures were elucidated by spectral analysis. Results: Seventeen compounds were isolated and identified as lasidiol p-methoxybenzoat (1), β-selinene (2), xanthatin (3), xanthinosin (4), luteone (5), daucosterol (6), 4β,5β-epoxy xanthatin-1α,4α-endoperoxide (7), (6S,9R)-vomifoliol (8), dehydrovomifoliol (9), 3(Z)-hexenyl-β-D-glycoside (10), 4-oxo-bedfordia acid (11), 11α,13-dihydro-8-epi-xanfbut (12), scopolin (13), pinoresinol (14), β-sitosterol (15), quercetin (16), and methyl p-hydroxybenzonate (17). Conclusion: The compounds 2, 8, 9, 10, and 17 are isolated from the plants of Xanthium L. for the first time and the compounds 1, 5, 7, 11 and 13 are isolated from the whole herbs of X. mongolicum for the first time.

Metabolism profile of sulfation of chrysin in human small intestine S9 / 中国生化药物杂志

Objective To investigate the metabolic profile of chrysin in SULT1A3 and human small intestine S9.Methods After incubation of chrysin using in vitro SULT1A3 and human small intestine S9 system, high-performance liquid chromatography was utilized to determine the sulfates of chrysin.Mass spectrum(MS) were employed to elucidate the structures of metabolite.Results In the SULT1A3 with PAPS, Km were (3.06 ±1.04) and (0.41±0.06) μM, Vmax were (12.13 ±1.30) and (6.72 ±1.61) nmol/(min· mg), Vmax/Km were 3.96 and 16.39 mL/(min· mg), respectively.In the human small intestine S9 with PAPS, Km were (1.92 ±0.35) and (0.01 ±0.00) μM, Vmax were (0.52 ±0.02) and (0.08 ± 0.02) nmol/(min· mg), Vmax/Km were 0.27 and 8.00 mL/(min· mg).The metabolic behavior of chrysin in SULT1A3 and human small intestine S9 both were followed biphasic kinetics.The sulfation of chrysin in SULT1A3 showed a significant correlation with that in human small intestine S9(R2 =0.985).Conclusion The result indicates that SULT1A3 is the major enzyme to the metabolism of chrysin, human small intestine may be the main metabolic organs of chrysin.

The characteristic analysis on intestinal metabolism of Pueraria lobata (Willd.)Ohwi / 国际中医中药杂志

Objective To establish a method to compare the difference of exposure component of oral taking different part ofPueraria Lobata (Willd.) Ohwi in intestine.Methods After the incubation of puerarin,extraction Radix Puerariae and Flos Puerariae with S9,the mixtures were centrifuged to get the supernatant for analysis with HPLC.Results The research showed that the metabolic rate of puerarin was higher than that of extractions because of the concentration of enzyme.The difference between the fingerprints of Radix Puerariae and Flos Puerariae Lobata indicated that there was a difference of chemical components in such two parts of Kudzuvine Root.Conclusion The profile of intestinal metabolism can be revealed in the research of gut wall metabolism in the course of intestinal absorption by S9 incubation.

Chemical constituents of Euphorbia royleana / 中草药

Objective: To study the chemical constituents in the aerial parts of Euphorbia royleana. Methods: The constituents were isolated and purified by chromatographic of silica gel, Sephadex LH-20, RP18, and MCI columns, and their structures were elucidated by spectroscopic analyses. Results: Twelve compounds were isolated from the 70% acetone extracts in the aerial parts of E. royleana and their structures were identified as (6S, 9R)-roseoside (1), 13-carboxyblumenol C 9-O-β-glucoside (2), 3, 3'-dimethylellagic acid-4-O-β-D-glucopyranoside (3), cycloart-23-ene-3β, 25-diol (4), 23(E)-25-methoxycycloart-23-en-3β-ol (5), α-amyrin (6), triptohypol F (7), 9(11), 12-dieneoleana-3β-ol (8), friedelane-3β, 29-diol (9), D:A-friedoolean-29-ol-3-one (10), dischidiol (11), and lupeol (12). Conclusion: Compounds 1, 2, 7-11 are obtained from the plants in Euphorbia L. for the first time and compounds 3-5 and 12 are isolated from this plant for the first time.

Study on sulfation metabolism of scutellarein in FVB/NCrIVr mice / 中国药学杂志

OBJECTIVE: To investigate the characteristics of sulfation of scutellarein in FVB/NCrIVr (FVB) mice. METHODS: FVB mouse intestinal perfusion model and incubation system with FVB mouse liver S9 fractions were adapted to conduct the study. HPLC-MS/MS and HPLC-UV were used to identify and quantify scutellarein and its metabolites in the samples. RESULTS: One sulfation metabolite and one glucuronidation metabolite were detected in the small intestinal perfusate. There was no significant difference between the excretion rates of sulfation metabolite and glucuronidation metabolite in small intestinal perfusate (P=0.435), while only sulfation metabolite of scutellarein could be detected in colon perfusate, scutellarein, sulfation metabolite and glucuronidation metabolite of scutellarein could all be detected in biliary samples, indicating an entero-hepatic circulation of scutellarein. In the liver S9 fractions, sulfation rate at 20 μmol·L-1 was sig nificantly higher than those at 10 and 40 μmol·L-1 (P<0.05). CONCLUSION: Sulfation was found to be the most important metabolism route in the intestinal disposition of scutellarein. There is probably a substrate inhibition effect in the sulfation of scutellarein in liver S9 fractions.

The Study on Synthesis on 20(S)-9-nitrocamptothecin / 医学研究杂志

20(S)-9-nitrocamptothecin was synthesized from camptothecin by nitration reaction using mixed nitrate salts in sulfuric acid medium.The yield was raised from 31% to 40% after chromatographic purification treatment.The best reaction conditions was as follows:0.5 g of camptothecin;30ml of sulfuric acid;0.007mol of nitrate salts(NH4NO3/CaNO3=1/1).The reaction was completed in 24h at 20℃.The advantage of this method is high yield and easily purification.

A Novel In Vitro Method for the Metabolism Studies of Radiotracers Using Mouse Liver S9 Fraction / 대한핵의학회잡지

PURPOSE: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of 18F-labeled radiotracers. MATERIALS AND METHODS: Mouse liver S9 fraction was isolated at an early step in the course of microsome preparation. The in vitro metabolism studies were carried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at 37 degrees C, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. RESULTS: The radiotracer [18F]1 underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer [18F]2 was metabolized to three metabolites including 4-[18F]fluorobenzoic acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. CONCLUSION: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.

Phosphorylation of Glycogen Synthase Kinase-3beta(S9) Induced Neuronal Cell Survival by Sulindac in the Rat Retina

PURPOSE: Inactivation of glycogen synthase kinase-3beta (GSK-3beta) by S9 phosphorylation is implicated in neuronal cell survival. In this study, we examined the involvement of GSK-3betaS9) phosphorylation on retina cell survival by sulindac (SLD) in model of retina ischemia. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 60 minutes in adult rats. SLD was treated pre and after (0.01 to 0.1 mM) ischemic injury. In vitro study, the retinas were isolated at postnatal 1-2day and were used to glutamate for ischemic injury. For morphological study, retinas were embedded in resin 24 hours after ischemic injury. The patterns of retinal cell were determined using light microscopy. Western blot analysis was performed using GSK-3beta(S9) and phospho-GSK-3beta(S9) antibodies. RESULTS: In ischemic animal model, cell death with necrosis and apoptosis was observed, treatment with SLD was reduced cell death. In vitro study, treatment of glutamate were reduce dose dependent manners, SLD treatment were decrease retina cell death. Western blot analysis of GSK-3beta(S9) phosphorylation known to induce neuronal cell survival, were increased in the SLD treated retina in ischemic injury. CONCLUSIONS: This study suggest that GSK-3beta(S9) is one of the effect by which SLD treatment protect retina from neuronal cell death.

The Effect of Bisphenol A and Dibutyphthlate Treated With Rat Liver S9 on Proliferation of MCF-7 Cells / 环境与健康杂志

Objective To study the effect of bisphenol A (BPA) and dibutyphthlate (DBP) treated with Rat Liver S9 on proliferation MCF-7 cells. Methods Added the positive control 17?-estradiol (E2) and compounds of BPA and DBP with the effect of Rat Liver S9 to MCF-7 cells proliferation respectively and determinated the quantity of MCF-7 cells with MTT assay. Results The activity of E2 and DBP stimulated MCF-7 cells decreasing significantly with the effect of Liver S9 than without the effect of Liver S9, while BPA stimulated MCF-7 cells increasing significantly with the effect of Liver S9 than that without the effect of Liver S9. Conclusion E2 and DBP have a lower estrogenic activity with the effect of S9 than without the effect of S9. BPA shows a higher estrogenic activity with the effect of S9 than without the effect of S9.

Molecular interactions between ribosomal proteins—An analysis of S7–S9, S7–S19, S9–S19 and S7–S9–S19 interactions.

Ribosomal proteins S7, S9 and S 19 from Escherichia coli have been studied by the sedimentation equilibrium technique for possible intermolecular interaction between pairs of proteins as well as in a mixture of 3 proteins. The proteins were isolated to a purity greater than 95% and were characterized in the reconstitution buffer. It was observed that none of the proteins has a tendency to self-associate in the concentration range studied in the temperature range 3–6°C. Protein S9 behaves differently in the presence of other proteins. Analysis of the sedimentation equilibrium data for S7–S9, S9– S19 and S7–S9–S19 complexes revealed the need for considering the presence of a component of higher molecular weight in the system along with the monomers and their complexes to provide a meaningful curve-fitting of the data. Proteins S7 and S19 were found to interact with an equilibrium constant of association of 3 ± 2 × 104 M–1 at 3°C with a Gibbs free energy of interaction ΔG° of -5·7 kcal/mol. These data are useful for the consideration of the stabilization of the 30S subunit through protein-protein interactions and also help in building a topographical model of the proteins of the small subunit from an energetics point of view.