Production and application of 3-phosphoadenosine-5- phosphosulfate / 生物工程学报

Sulfated compounds are widely present in cytoplasm, on cell surface, and in extracellular matrix. These compounds play important roles in cell development, differentiation, immune response, detoxication, and cell signal transduction. 3-Phosphoadenosine-5-phosphosulfate (PAPS) is the universal sulfate group donor for the biosynthesis of sulfated compounds. Up to now, the synthesis of PAPS is still too expensive for industrial applications. This review focuses on the recent progress of PAPS production and summaries the application of PAPS, particularly in the production of glucosinolate, heparin, condroitin sulfate, and oxamniquine production.

Effect of Saposhnikovan Sulphate on the Recruitment and Re-education of Macrophages by MCF-7 Cells In Vitro through CCL3 / 中国药学杂志

OBJECTIVE: To observe the inhibition of saposhnikovan sulphate obtained by ultrasonic extraction(S-USPS) on human leukemia MCF-7 cells in vitro and the effect of S-USPS on the recruitment and re-education of macrophages by MCF-7 cells. METHODS: S-USPS was obtained from saposknikovan(SPS) by the method of chlorosulfonic acid-pyridine complex. The inhibition of S-USPS and USPS on proliferation of MCF-7 cells was measured by MTT. THP-1-derived macrophages were differentiated in vitro. The effect of S-USPS on the recruitment of macrophages by MCF-7 cells was detected by transwell migration assay. The effects of S-USPS on the expression and secretion of chemokine were detected by qRT-PCR and ELISA. The effects of S-USPS and CCL3 on the expression of inflammatory cytokines were detected by qRT-PCR. RESULTS: The inhibitory rate of S-USPS on the proliferation of MCF-7 was dose-dependent and time-dependent and was higher than that of USPS(P<0.05).S-USPS increased the transcription and secretion of CCL3 by MCF-7 cells. There were higher expressions of IL-1β, IL-6, and TNF-α mRNA and lower expressions of TGF-β, IL-10 mRNA of macrophages in the S-USPS group than in the control group and MCF-7 supernatant group(P<0.05). The promotion of S-USPS on the expressions of IL-1β and TNF-α was inhibited by CCL3 neutralizing antibody(P<0.05). CONCLUSION: There are significant evidences that the saposhnikovan sulphate effectively promotes the recruitment of macrophages by MCF-7 cells in vitro, and re-educates their secretion profile toward M1-type.

Study on metabolic pathways of salidroside in rats / 中草药

Objective: To analyze the metabolic pathways of salidroside when gavaged to rats, and to identify its in vivo metabolites. Methods: A UPLC-Q-TOF-MS method was established and applied to identify the metabolites of salidroside in bile, urine, feces, and plasma samples after ig administration of salidroside 200 mg/kg to rats. Results: A total of 20 metabolites were characterized in rats. The dominant metabolic pathways of salidroside include glucuronidation, acetylation, sulfation, and methylation. Conclusion: Salidroside undergoes extensive phases I and II metabolism in rats. The main excretion path is the phase II conjugated metabolites via urine, feces, and bile. The parent drug of salidroside is the main exposure material in plasma.

Research Progress in the Antitumor Effects of Sulfated Polysaccharides / 中国药师

Sulfated polysaccharides are with hydroxyl group containing sulfate radicals, which can be obtained by natural extraction or chemical modification of sulfation. Recent studies have shown that sulfated polysaccharides exhibit different levels of antitumor activ-ity, especially in comparison with those before chemical modification; therefore, they have attracted wide attention. This article briefly reviewed the domestic and foreign researches on the antitumor effects of natural sulfated polysaccharides and chemical synthesized sul-fated polysaccharides in recent years.

Arenobufagin is a novel isoform-specific probe for sensing human sulfotransferase 2A1

Human cytosolic sulfotransferase 2A1 (SULT2A1) is an important phase II metabolic enzyme. The detection of SULT2A1 is helpful for the functional characterization of SULT2A1 and diagnosis of its related diseases. However, due to the overlapping substrate specificity among members of the sulfotransferase family, it is difficult to develop a probe substrate for selective detection of SULT2A1. In the present study, through characterization of the sulfation of series of bufadienolides, arenobufagin (AB) was proved as a potential probe substrate for SULT2A1 with high sensitivity and specificity. Subsequently, the sulfation of AB was characterized by experimental and molecular docking studies. The sulfate-conjugated metabolite was identified as AB-3-sulfate. The sulfation of AB displayed a high selectivity for SULT2A1 which was confirmed by reaction phenotyping assays. The sulfation of AB by human liver cytosols and recombinant SULT2A1 both obeyed Michaelis-Menten kinetics, with similar kinetic parameters. Molecular docking was performed to understand the interaction between AB and SULT2A1, in which the lack of interaction with Met-137 and Tyr-238 of SULT2A1 made it possible to eliminate substrate inhibition of AB sulfation. Finally, the probe was successfully used to determine the activity of SULT2A1 and its isoenzymes in tissue preparations of human and laboratory animals.

In vitro inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed Caulerpa racemosa (Caulerpaceae, Bryopsidales) / Inativação in vitro da geração de trombina por frações polissulfatadas isoladas da macroalga verde cenocítica tropical Caulerpa racemosa (Caulerpaceae; Bryopsidales)

Pharmacological efficacy of Caulerpa racemosa (Chlorophyta) sulfated polysaccharidic (SPs) fractions (F I→III) on models of coagulation and inflammation has been demonstrated, but not their effects on thrombin generation (TG). This study examined fractions for composition and physical-chemical characteristics and in vitro inactivation of TG by F I and F II in 60-fold diluted human plasma using continuous method. Papain-extraction yield of 0.7% revealed F I→III by DEAE-cellulose chromatography, with differences among the relative proportions of sulfate (17.37-24.00%), total sugars (30.03-48.34%) and absence of proteins. Charge density patterns and molecular sizes > 100 kDa of the fractions were verified by both agarose/polyacrylamide analyses, respectively. These electrophoreses combined with toluidine blue/Stains-All also indicated nonSPs. Anticoagulant effects of 4.76 (F I), 12.00 (F II) and 2.32 (F II) IU mg-1 by activated partial thromboplastin time test were recorded against heparin (193 IU mg-1), without changes in prothrombin time. Diluted plasma treated with F I and F II reduced concentration-dependent and sulfation pattern TG by both intrinsic and extrinsic pathways, with 50% inactivation by intrinsic pathway of F II even at 4.1 µg. Heparin abolished TG at least 4-fold lower. Therefore, C. racemosa produces SPs with TG inhibition.

Eficácia farmacológica de frações (F I→III) polissacarídicas sulfatadas (PSs) da Chlorophyta Caulerpa racemosa sobre modelos de coagulação e inflamação tem sido demonstrada, exceto seus efeitos sobre geração de trombina (GT). Examinaram-se frações quanto à composição, características físico-químicas e inativação in vitro de GT por F I e F II, em plasma humano diluído 60 vezes usando método contínuo. Rendimento de extração-papaína (0,7%) revelou, por cromatografia de DEAE -celulose, F I→III com diferenças entre as proporções relativas de sulfato (17,37-24,00%), açúcares totais (30,03-48,34%) e ausência de proteínas. Foram verificados, por ambas as análises agarose/poliacrilamida, graus de densidade de carga e tamanhos moleculares > 100 kDa das frações, respectivamente. Também essas eletroforeses, combinadas com azul de toluidina/Stains-All, indicaram polissacarídeos não sulfatados. Foram registrados, pelo teste do tempo de tromboplastina parcial ativada, efeitos anticoagulantes de 4,76 (F I), 12,00 (F II) e 2,32 (F II) UI mg-1 contra heparina (193 UI mg- 1), porém não modificando tempo de protrombina. Plasma diluído tratado com F I e F II reduziu GT por ambas as vias intrinsíca/extrínsica, dependente de concentração e grau de sulfatação, com F II em 4,1 µg apresentando eficácia de 50% pela via intrínsica. Heparina, quatro vezes menos, aboliu GT. Portanto, C. racemosa produz PSs com inibição de GT.

Optimization of sulfated modification conditions of polysaccharide from Bupleurum Chinense and comparison of their antioxidant abilities between before and after modification / 吉林大学学报(医学版)

Objective: To investigate the influencing factors of sulfated modification of Bupleurum chinense polysaccharides (BCP),and to elucidate the possible mechanism of improving the antioxidant ability of sulfated BCP (S-BCP).Methods:BCP was sulfated by chlorosulfonic acid-pyridine method.The degree of substitution (DS)of S-BCP was observed by adjusting the volume ratios of chlorosulfonic acid to pyridine (1:2,1:4,and 1:8).The structures of BCP and S-BCP were analyzed by infrared (IR)spectroscopy,the morphology of BCP and S-BCP were observed under scanning electron microscopy (SEM).The antioxidant model was established by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)free radical scavenging.The experiment was divided into positive control group,BCP group and S-BCP group,and the scavenging rates of DPPH free radical in various groups were compared. Results:When the volume ratio of chlorosulfonic acid to pyridine was 1 : 4,the reaction time was 2 h and the reaction temperature was 60 ℃,the maximum sulfur content percentage of S-BCP was 18.62% and the DS was the highest (DS = 2.32 ).Compared with BCP group, the scavenging rate of DPPH free radical of S-BCP was significantly increased (P <0.05).Conclusion:The volume ratio of chlorosulfonic acid to pyridine can affect the DS of S-BCP.The sulfated modification can increase the anti-oxidant capacity of BCP by changing its physic-chemical characters and spatial conformation.

Protective effect of Ganodermalucidum polysaccharide sulfate on cerebral ischemia reperfusion injury in rats and its mechanism / 吉林大学学报(医学版)

Objective:To modify Ganodermalucidum polysaccharides(GLP) with sulfate and observe the protective effect of Ganodermalucidum polysaccharide sulfate (GLPS) on the cerebral ischemia reperfusion injury in the rats,and to investigate its mechanism.Methods:GLP was modified by sulfation to obtain GLPS.A total of 100 SD rats were randomly divided into sham operation group, model group, GLP group (40 mg·kg-1·d-1), GLPS group (40 mg·kg-1·d-1) and nimodipine group (1 mg·kg-1·d-1).The cerebral ischemia reperfusion models were established by middle cerebral artery occlusion method in the rats.The neurologic deficit score and the content of water in brain tissue of the rats with cerebral ischemia reperfusion injury were detected and the activities of superoxide dismutase(SOD) and the levels of malondialdehyde (MDA) were detected.The levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) in the brain tissue homogenate were detected by ELISA.Western blotting method was used to detect the protein expression levels of HSP70 and p-Akt in the brain tissue of the rats.Results:Compared with model group, the neurological function scores of the rats in GLP group and GLPS group were decreased(P<0.01),the water contents in brain tissue were decreased(P<0.05), the SOD activities were increased and the MDA levels were decreased(P<0.05), and the levels of NF-κB, TNF-α, IL-1 and IL-6 were decreased(P<0.05);the effect in GLPS group was significantly better than that in GLP group(P<0.05).The results of Western blotting method showed that the p-Akt protein expression levels in the brain tissue of the rats in GLP and GLPS groups were increased compared with model group (P<0.05);compared with model group, the HSP-70 protein expression level in the brain tissue of the rats in GLPS group was increased(P<0.01),but the effect in GLP group was not obvious.Conclusion:Sulfation can significantly improve the protective effect of GLP on the cerebral ischemia reperfusion injury in the rats and its mechanism may be related to regulating the HSP70/PI3K/Akt signaling pathway and inhibiting the inflammatory reaction damage to the nerve cells of reperfusion.

Sulfated modification and DPPH free radical scavenging ability of polysaccharide from Rhodiola sachalinensis / 中草药

Objective: To optimize the sulfated modification conditions of Rhodiola sachalinensis polysaccharide (RSP) and improve the anti-oxidant activity of RSP. Methods: RSP were sulfated by chlorosulfonic acid-pyridine method, and the optimal conditions for the sulfated modification were determined by single factor experiments. The physical and chemical properties of RSP and sulfated RSP (S-RSP) were analyzed by infrared spectroscopy (IR) and scanning electron microscopy (SEM). The relation between substituting degree (DS) of S-RSP and anti-oxidative activity of polysaccharide was investigated by testing the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability of RSP and S-RSP. Results: When the volume ratio of chlorosulfonic acid to pyridine was 1∶4, the reaction time was 2 h and the reaction temperature was 60 ℃, the maximum sulfur content of S-RSP was 18.83% and the DS was the highest for 2.38. Moreover, the anti-oxidant activity of RSP was enhanced by the sulfated modification, and there was a certain positive proportional relationship between DS and DPPH free radical scavenging ability of S-RSP. Conclusion: The volume ratio of chlorosulfonic acid to pyridine affects the DS of S-RSP, and the sulfated modification can increase the anti-oxidant capacity of RSP by changing its polarity.

Study on kinectics characteristics of the sulfation of apigenin by SULTIA3 / 中国生化药物杂志

Objective To investigate the kinectics characteristics of sulfation of apigenin mediated by SULTIA3.Methods After incubation of apigenin using in vitro SULT1A3 system, high-performance liquid chromatography was utilized to determine the sulfates of apigenin.Mass spectrum(MS) were employed to elucidate the structure of metabolite.The program GraphPad Prism 5 was used to perform the kinetic characterization of SULT1A3 catalyzed metabolism of apigenin.Results A liner calibration curve for the assay of apigenin was validated in the range of 0.15625 ~30 μM with the recoveries of at least 80% and intra-day and inter-day RSD of less than 15%.Metabolic product of apigenin and SULT1A3 in the incubated system was identified one monosulfate.The metabolic behavior of apigenin in SULT1A3 was followed substrate inhibition kinetics.Apparent kinetic parameters of metabolism of apigenin by SULT1A3, Kmwas(0.355 ±1.04) μM and Ksi was(23.62 ±0.06) μM,Vmax was(65.71 ±1.30) nmol/(min? mg),Vmax/Km was 185.10 mL/(min? mg).Conclusion SULT1A3 can mediate the binding of apigenin sulfonated reaction, and the character of enzymatic kinetics shows substrate inhibition.Sulfation of apigenin mediated by SULTIA3 may play an important role in phaseⅡmetabolic in vivo.

Glycosylation and sulfation of emodin by Gliocladium deliquescens NRRL 1086 / 中国天然药物

The present study was designed to explore the substrate scope and biocatalytic capability of Gliocladium deliquescens NRRL 1086 on phenolic natural products. Emodin was subjected to the fermentation culture of Gliocladium deliquescens NRRL 1086 according to the standard two-stage protocol. The biotransformation process was monitored by HPLC-DAD-MS, the main product was isolated by column chromatography, and the structure was elucidated on the basis of NMR spectroscopy. Emodin could be fully metabolized by Gliocladium deliquescens NRRL 1086, resulting in high yield of emodin 6-O-β-D-glucopyranoside and small amount of sulfated product. In conclusion, our results may provide a convenient method to prepare emodin 6-O-β-D-glucopyranoside and the microbe catalyzed glucosylation/sulfation will give an inspiration to pharmacokinetic model studies in vitro.

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.

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.

Atividade anti-HSV-1 in vitro de galactomanana quimicamente sulfatada de sementes de Leucaena leucocephala / In vitro anti-HSV-1 activity of a chemically sulfated galactomannan from Leucaena leucocephala seeds

Obteve-se uma galactomanana quimicamente sulfatada (LLS-2) a partir de polissacarídeo extraído de sementes de Leucaena leucocephala, a qual apresentou 15.2% de sulfato e grau de derivatização de 0,60, e, seu efeito antiviral sobre a replicação do vírus Herpes simplex tipo 1 (HSV-1) em células Vero foi avaliado pela metodologia de redução do número de unidades formadoras de placas. LLS-2 apresentou 93.7% de inibição da replicação viral à concentração de 2,5 ?g/ml, quando adicionado durante as etapas iniciais de replicação, com um índice de seletividade maior que 1.000, sugerindo que LLS-2 inibe a ligação de HSV-1 às células hospedeiras.

A galactomannan extracted from the seeds of Leucaena leucocephala was sulfated chemically, yielding a polymer (LLS-2) with 15.2% sulfate by weight (degree of sulfation 0.60), and its effect on the replication of Herpes simplex virus 1 (HSV-1) in Vero cells was investigated. When added during infection and early replication, LLS-2 showed 93.7% inhibition of HSV-1 replication at a concentration of 2.5 ?g/mL, according to the reduction in the number of viral plaques, and a selectivity index higher than 1,000, suggesting that it inhibits HSV-1 binding to the host cell.

Modificación y aaracterización de una vermiculia Colombiana con especies de tiitanio, zirconio y sulfato / Modifiction and characterization of a Colombian vermiclite with titannium, zirconium and sulfat / Modificção e carracterização do vermiculite Colombian com titânio, zirconiõ e sulfato

El presente trabajo reporta los resultados de la caracterización química, estructural y textural de una vermiculita de origen colombiano modificada con especies simples de Zr y Ti, y el sistema mixto Ti-Zr en relaciones atómicas nominales Zr/(Ti + Zr) = 0,1 y 0,5. Adicionalmente, se evaluó la incidencia de la presencia de sulfato sobre los sistemas mencionados bajo las relaciones nominales sulfato/metal 0; 0,25 y 0,35, incorporando el sulfato ya sea en la solución intercalante, o por impregnación vía húmeda sobre los sólidos modificados y calcinados. Los resultados indican que cuando la sulfatación se lleva a cabo por intercalación, la naturaleza química de la solución intercalante afecta notoriamente la cantidad de metal incorporado, dependiendo de la relación sulfato/metal empleada. De otro lado se estableció que, cuando se lleva a cabo la sulfatación por impregnación, las propiedades texturales, estructurales y morfológicas de los sólidos obtenidos se ven fuertemente afectadas y estan acompañadas de una fijación de azufre significativamente mayor que para los sólidos sulfatados por intercalación.

The present work reports the results of chemical, structural and textural characterization of a Colombian vermiculite modified with simple species of Zr and Ti, and the mixed system of Ti-Zr in nominal atomic relations Zr/(Ti + Zr) = 0,1 and 0,5. Additionally, the incidence of sulfate presence was evaluated under the nominal relations sulfate/metal 0; 0,25 and 0,35, incorporating sulfate either in the intercalation solution, or by impregnation on modified and calcined solids. The results indicate that when the sulfation is carried out by intercalation, the chemical nature of the intercalation solution affects the amount of incorporated metal, depending on the relation used sulfate/metal. When the sulfation by impregnating is carried out, the textural, structural and morphologic properties of the obtained solids are strongly affected and are accompanied of a sulfur fixation significantly greater than for solids sulfated by intercalation.

O trabalho atual relata os resultados do produto químico, caracterização estrutural e textural de um vermiculite Colombian modificado com espécie simples do Zr e o Ti, e o sistema misturado do Ti-Zr no Zr/atômico nominal das relações (Ti + Zr) = 0.1 e 0.5. Adicionalmente, a incidência da presença do sulfato foi avaliada sobre o sulfato das relações/metal nominais 0; 0.25 e 0.35, incorporando sulfato já seja na solução ou do intercalação, ou pelo impregnação em sólidos modificados e calcinados. Os resultados indicam que quando o sulfatação é realizado pelo intercalação, a natureza química da solução do intercalação afeta a quantidade de metal incorporado, dependendo do sulfato/metal usados na relação. Quando o sulfação impregnando é realizado, as propriedades texturaes, as estruturais e as morfologicas de sólidos obtidos fortemente estão afetadas e acompanhadas de uma fixation de enxôfre significativamente mais grande do que para os sólidos sulfatados pelo intercalação.

Sulfation techniques of Bletilla striata polysaccharide by orthogonal design / 中草药

Objective To optimize the sulfation-conditions of Bletilla striata polysaccharide (BSPS). Methods BSPS was sulfated with chlorsulfonic acid-pyridine. Reagent proportion, reaction temperature, and reaction time were selected with substitution degree and yield as index by L_9(3~4) orthogonal design. Results Results showed that the condition with 6∶1 volume ratio of pyridine and chlorine sulfonic acid, reaction temperature of 85 ℃, reaction time of 2 h was the optimum. Conclusion The optimal method for sulfation of BSPS is feasible, and it provides valuable parameters for further enlarged test.