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[The effects of the polysaccharides from Dermatocarpon miniatum on oxygen radicals and lipid peroxidation].

OBJECTIVE: To study the effects of Dermatocarpon miniatum (DEM) polysaccharides scavenging oxygen radicals and inhibiting lipid peroxidation. METHODS: .OH was produced by Fenton reaction and O2-. was produced by the oxidation of pyrogallol. The inhibition to LPO was determined by the colorimetry for testing the relative content of MDA. RESULTS: DEM polysaccharides could scavenge oxygen radical. The amounts of scavenging 50% (EC50) .OH was 1.72 mg/ml, the EC50 for O2-. was 2.12 mg/ml. DEM polysaccharides could also decrease the content of MDA. CONCLUSION: DEM polysaccharides has the effects of scavenging oxygen radicals and the inhibition to LPO. Its activities showed positive correlation with the amounts.

Antioxidants↗

[A study of scavenging action of purslane aquenous extracts on oxygen free radical].

Auto-oxidation in alkalesent solution with the help of pyrogallol can generate O2.-, and the system of 1, 10-phenathroline-Fe(II)/H2O2 can produce .OH. Spectropholometry (wave length 322 nm and 536 nm) was used to observe the effect of fresh purslane aqueous extracts and the purslane aquenous extracts processed by boiling water upon O2.- and .OH. The results showed that fresh purslane aqueous extracts could obviously help generate O2.- and reduce .OH, and the aqueous extracts of purslane processed by boiling water had a scavenging action on O2.- and .OH greatly.

Drugs, Chinese Herbal↗

Cloning and Expression of Targeting CuZn-SOD to Central Nervous System.

Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders. Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases. The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively. The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody. By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system. So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.

Journal Article↗

[Studies on dynamic changes of SOD and MDA in cultured cells from Schistosoma japonicum schistosomula].

AIM: To observe the dynamic changes of superoxide dismutase(SOD) and maldondialdehyde(MDA) in cultured cells from Schistosoma japonicum schistosomula and to explore the degeneration process of cultured cells. METHODS: Pyrogallol method and thiobarbituric acid method were used to determinate the levels of SOD and MDA, respectively, in the cells cultured for 0-54 days. RESULTS: SOD in the cells reached a peak level on d12 of the early stage of culture and then decreased. The MDA levels in the cultures cells ascended gradually, and had two peaks on d18 and d48, during the period of culture. CONCLUSION: The cultured cells from S. japonicum schistosomula degenerated progressively.

Animals↗

[Scavenging effects of Astragalus and Gynostemma pentaphyllum with its product on O2-. and .OH].

The scavenging effects of Astragalus and Gynostemma pentaphyllum with its products on O2-. (produced by the autoxidation of pyrogallol) and .OH (generated from the Fenton reaction) were investigated by electrochemical method. The results showed that they could scavenge the active oxygen free radicals effectively, however, their scavenge ability was different for O2-. and .OH. The inhibitory effect of Ginsenoside Rb1 and Astragaloside IV on O2-. and .OH was the most strongest. Their IC50 values were calculated respectively. The scavenging ability of Astragalus samples on O2-. was in the order of the following: astragaloside IV > crude radix astragali > mongolia radix astragali > hebei radix astragali > radix astragali > radix astragali mixfry with honey; and the scavenging effect on .OH was: astragaloside IV > hebei radix astragali > radix astragali mix-fry with honey > radix astragali > crude radix astragali > mongolia radix astragali. The scavenging activity of gynostemma samples on O2-. was: ginsenoside Rb1 > gynostemma jujube tea > tabellae Jiaogulanosidi > anguo market gynostemma > hebei qingxian gynostemma; while on .OH, the inhibitory ability was: ginsenoside Rb1 > gynostemma jujube tea > anguo market gynostemma > hebei qingxian gynostemma > tabellae Jiaogulanosidi.

Astragalus Plant↗

Anti-inflammatory actions of flavonoids and structural requirements for new design.

Flavonoids are low molecular weight compounds rich in seeds, citrus fruits, olive oil, tea and red wine, with potent antioxidant, cytoprotective and antiinflammatory activities. Flavonoids are composed of a three-ring structure (A,B and C) with various substitutions; they can be subdivided according to the presence of an oxy group at position 4, a double bond between carbon atoms 2 and 3 or a hydroxyl group in position 3 of the C (middle) ring. Particular hydroxylation patterns of the B ring of the flavones permit them to inhibit histamine, tryptase, interleukin-6 and interleukin-8 release from human umbilical-cord derived cultured mast cells, as well as from macrophages. The catechol (o-dihydroxy) group in the B ring as in quercetin confers potent inhibitory ability, while a pyrogallol (trihydroxy) group, as in myricetin, produces even higher activity. However, addition of one hydroxyl group on position 2′ of the B ring, as in the flavonol morin, renders this compound inactive. The C2-C3 double bond of the C ring appears to increase scavenger activity because it confers stability to the phenoxy-radicals produced, while the 4-oxo (keto double bond at position 4 of the C ring) increases free radical scavenger activity by delocalizing electrons from the B ring. The 3-OH group on the C ring appears tobe critical for anti-inflammatory activity. Inhibition of mast cell secretion was shown to be mediated by a 78-kD phosphoprotein which has been cloned and serves as a bridge between the cell surface and the cytoskeleton. Phosphorylation at particular sites in the C-terminus unfolds the three dimensional structure of this protein making actin binding sites accessible; crosslinking with actin in the cytoskeleton prevents secretion of inflammatory mediators. These properties present unique opportunities for the synthesis of new compounds for the treatment of inflammatory and possibly proliferative disorders.

Journal Article↗

[The simultaneous determination of iron(III) and aluminium(III) in quartz sand by first derivative spectrophotometry].

It is found that in a buffer solution of HAc-NaAc (pH-6.0), iron (III) and aluminium (III) react with ethylene glycol diethy diaminoteracetic acid (EGTA), pyrogallol red (PR) and cetylpyridinium bromide (CPB) to form quarternary complex. Micro iron (III) and aluminium (III) can be simultaneous determined by the first derivative spectrophotometry of the quarternary complex. The RSD for the determinations of iron and aluminium are 4.5% and 1.1% respectively, and the recoveries are 93%-98% and 95%-102% respectively. This method has been applied to the determination of micro iron and aluminum in quartz sand with satisfactory results.

Aluminum↗

Total prevention of the development of in vitro tolerance to organic nitrates. Experiments with antioxidants.

The nearly total inhibition of development of pharmacological tolerance to an organic nitrate is reported here for the first time. The development of in vitro tolerance in the rabbit aorta to isosorbide-5-mononitrate (CAS 87-33-2) was potently inhibited by five structurally unrelated antioxidants--diaminodurol, ascorbic acid, potassium sulphite, pyrogallol and quercetin. Diaminodurol, ascorbic acid and potassium sulphite decreased, but quercetin increased, the spasmolytic activity of isosorbide-5-mononitrate. Diaminodurol, potassium sulphite, quercetin and ascorbic acid potently inhibited the spasmolytic activity of nitric oxide (NO). Quercetin also inhibited the development of in vitro tolerance to glyceryl trinitrate. It is suggested that tolerance to organic nitrates is the result of biochemical damage caused by a reactive intermediate such as NO. To test this possibility directly the effect of pretreatment with NO on the spasmolytic activity of glyceryl trinitrate (CAS 55-63-0) was examined. This pretreatment produced a small but significant tolerance to glyceryl trinitrate and to SIN-1 (3-morpholinosydnone imine), which also acts through guanylate cyclase. There was no effect on the activity of the unrelated vasodilators nitrendipine and theophylline. It is concluded that the reaction between NO and soluble quanylate cyclase is a real but minor cause of tolerance to organic nitrates. Other possible mechanisms of tolerance development are discussed.

Animals↗

[Studies on the chemical components of Rhodiola crenulata].

Six chemical components I-IV were isolated from the roots of Rhodiola crenulata (Crassulaceae). Five (I-V) of them were identified as known salidroside (I), p-tyrosol(II), pyrogallol, gallic acid, and beta-sitosterol, based on comparison of its Rf(TLC), mmp and spectral data with those of authentic samples. The sixth component was found to be a new delta 1-isopentenyl-3-O-beta-D-glucopyranoside and named crenulatin(VI). Its structure was confirmed on the basis of its spectral data (IR, MS, 1H- and 13C-NMR).

Coumarins↗

Inhibiting effects of Achyranthes bidentata polysaccharide and Lycium barbarum polysaccharide on nonenzyme glycation in D-galactose induced mouse aging model.

OBJECTIVE: To investigate the inhibiting effects and mechanism of achyranthes bidentata polysaccharide (ABP) and lycium barbarum polysaccharide (LBP) on nonenzyme glycation in D-galactose induced mouse aging model. METHODS: Serum AGE levels were determined by AGE-ELISA, MTT method was used to determine lymphocyte proliferation, IL-2 activity was determined by a bioassay method. Spontaneous motor activity was used to detect mouse's neuromuscular movement, latency of step-through method was used to examine learning and memory abilities of mouse, colormetric assay was used to determine hydroxyproline concentration in mouse skin, pyrogallol autoxidation method was used to determine superoxide dismutase (SOD) activity of erythrocytes. RESULTS: Decreased levels of serum AGE, hydroxyproline concentration in mouse skin and spontaneous motor activity in D-galactose mouse aging model were detected after treated with ABP or LBP, while lymphocyte proliferation and IL-2 activity, learning and memory abilities, SOD activity of erythrocytes, were enhanced. CONCLUSIONS: ABP and LBP could inhibit nonenzyme glycation in D-galactose induced mouse aging model in vivo and ABP has a better inhibiting effect than LBP.

Achyranthes↗

[The effects of banxia houpu decoction on a chronic mild stress model of depression].

OBJECTIVE: To study the effect of Banxia Houpu Decoction on a chronic mild stress model of depression and investigate the antidepressive mechanism. METHOD: With consumption of a 1% sucrose solution as an index, and by subjecting rats to a variety of mild stressors for a prolonged period of time, a chronic mild stress model was developed. The levels of the blood lipid were measured by blood lipid kits, the natural kill (NK) cell activity in the spleen was measured with the method of the enzyme lactate dehydrogenase (LDH) released, the superoxide dismutase (SOD) activity in red blood cell was assayed by the Autoxidation of Pyrogallol method, the NO synthase (NOS) activity in serum and tissue was measured by NOS kits, and the content of malondialdehyde(MDA) was measured by MDA kits. RESULT: Banxia Houpu Decoction significantly increased the consumption of sucrose solution, increased the level of the high density lipoprotein (HDL-C), decreased the level of the Triglyceride(TG) in serum, enhanced the activity of the NK in spleen, decreased the activity of the SOD in red blood and the activity of the NOS in serum and tissue, and reduced the content of MDA in tissue by effect on lipid Peroxidation in CNS model of depression. CONCLUSION: Banxia Houpu Decoction has antidepressant effect in different ways.

Animals↗

Artifactual oxidation of cholesterol during the analysis of cholesterol oxidation products: protective effect of antioxidants.

To study the influence of the addition of various antioxidants and their combinations on the artifactual oxidation of cholesterol during analysis, 2 factorial experiments were performed in duplicate. In the first experiment, 2 amounts of the following antioxidants were assayed: ethylenediaminetetraacetic acid (EDTA) disodium salt (0 and 1 mg), pyrogallol (0 and 600 microg), and butylated hydroxytoluene (BHT; 0 and 600 microg); in the second, EDTA disodium salt (0 and 1 mg), ascorbyl palmitate (0 and 600 microg), and BHT (0 and 600 microg). Under low oxidative conditions of dim light, evaporation of solvents at low temperatures, and cold saponification in darkness under nitrogen atmosphere, the addition of antioxidants showed no further protective effect. Furthermore, the presence of ascorbyl palmitate significantly increased the formation of cholesterol-5beta,6beta-epoxide, and 7beta-hydroxycholesterol.

Antioxidants↗

[Dynamic changes of enzymes activities and growth factors contents in human colostrum].

OBJECTIVE: To study the dynamic changes of the activities of superoxide dismutase (SOD), amylase (AM) and alkaline phosphatase (ALP) and of the contents of epithelium growth factors (EGF) and insulin growth factors (IGF-1) in human colostrum (lactation 1 - 7 days postpartum). METHODS: A total of 118 samples of human colostrum were taken from 20 healthy women postpartum. The activities of SOD, AM and ALP in human colostrum were determined by methods of pyrogallol autoxidation, colorimetry of iodine-starch and pyrocatechol phosphate respectively. EGF and IGF-1 contents in human colostrum were measured by radioimmunoassay. RESULTS: The SOD activity of human colostrum increased slightly during lactation 1 - 4 days postpartum, from (18.7 +/- 2.2) kU/L to (22.5 +/- 2.9) kU/L, then it tended to decrease, being (11.2 +/- 2.1) kU/L on the 7th day. The SOD activities in lactation 1 - 5 days postpartum were significantly higher than that on day 7 (P < 0.05 or P < 0.01). AM and ALP activities of human colostrum gradually decreased within a week postpartum. AM activity declined from (155 +/- 12) kU/L to (17 +/- 4) kU/L. ALP activity declined from (105 +/- 17) kU/L to (54 +/- 9) kU/L. AM activities in lactation 1 - 5 days postpartum were significantly higher than that on day 7 (P < 0.05 or P < 0.01); and ALP activity on the first lactation day was significantly higher than that on day 7 (P < 0.05). EGF and IGF-1 concentrations in human colostrum quickly declined during lactation 1 - 4 days postpartum. EGF concentration decreased from (105 +/- 11) micro g/L to (40 +/- 5) micro g/L, and IGF-1 concentration decreased from (25.9 +/- 2.7) micro g/L to (5.6 +/- 1.3) micro g/L. Both concentrations altered slightly during lactation 4 - 7 days postpartum. EGF concentrations in lactation 1 - 3 days postpartum were remarkably higher than that on day 7 (P < 0.05 or P < 0.01); IGF-1 concentration on the first lactation day was significantly higher than that on day 7 (P < 0.01). CONCLUSIONS: Human colostrum contains SOD, AM and ALP with high activities, and is also rich in EGF and IGF-1. All of these are indispensable to normal growth and development of infants. Breast-feeding should be emphasized and encouraged for infants.

Adult↗

[Spectrophotomatric determination of antioxidative activity of extractives of Chinese traditional medicine by supercritical fluid CO2 extraction technology].

Applying supercritical CO2 fluid extraction technology, flavonoids were extracted from Chinese traditional medicine asarum heterotropoides, atractylodes macrocephala, and rheum palmatun. Using the method of autoxidation pyrogallol (known as 325 nm method), the superoxide radical scavenging effect of the extraction was carried out in the buffer solution of HCl-tris (pH 8.2). With spectrophotometry, hydroxyl radical created by the system Co2+ + H2O2 in the reaction like Fenton reaction was eliminated by alizarin violet as the color developing agent in the buffer solution HCl-tris (pH 9.0) and the reaction condition was investigated. Result showed that these extractions are elimination agent for these radicals. Asarum heterotropoides is the best of the three.

Anthraquinones↗

Superoxide dismutase activity and expression in human venous and arterial bypass graft vessels.

UNLABELLED: Venous bypass grafts are more prone to accelerated atherosclerosis than arterial grafts, which is partly related to increased oxidative stress and diminished nitric oxide bioavailability. In veins superoxide production is dependent primarily on nox2 NAD(P)H oxidase expression, while in arteries nox4 appears to play an important role. This may in part explain differences in susceptibility to graft failure. Net levels of oxidative stress are however determined in parallel by the production as well as by degradation of free radicals (eg. by superoxide dismutases, catalases, thioredoxins etc). The differences in superoxide dismutase (SOD) expression and activity in human bypass conduit vessels remain unclear. Accordingly, we aimed to compare SOD activity and protein levels as well as its functional effects on superoxide production in segments of human internal mammary arteries (IMA) and saphenous veins (HSV) from patients undergoing bypass graft surgery (n=24). SOD activity was assessed by inhibition of pyrogallol autoxidation, Cu-Zn SOD and Mn SOD protein levels were studied by immunoblotting. Basal superoxide release was detected by lucigenin (5 microM) enhanced chemiluminescence. Total SOD activity did not differ significantly between HSV and IMA. Similarly, no difference was observed in SOD activity in the presence of KCN (Mn-SOD). Human bypass conduit vessels show amounts of Cu-Zn SOD or Mn-SOD protein levels. In both HSV and IMA segments superoxide production was more than doubled in the presence of SOD inhibitor-DETC. CONCLUSIONS: These studies suggest that the differences in oxidative stress between human arteries and veins are unlikely to be caused by SOD activity. However SOD plays and important role in amelioration of oxidative stress in both types of vessels.

Coronary Artery Bypass↗

[Sited-directed mutagenesis of hCu, Zn-SOD gene and its expression in Synechococcus sp. PCC7942].

The Cys111 genetic code of human copper/zinc superoxide dismutase (hCu, Zn-SOD) gene in the pESOD plamid was mutated into the Ala111 code with site-directed mutagenesis, and then the plamid pESODT111 which contained groESL promoter, mutated hCu, Zn-SOD gene, rbcS-polyA terminator and reporter gene (Kanr) was constructed and transduced into Synechococcus sp. PCC7942 with homologous recombination platform. The results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell. SDS-PAGE, Western blot and Pyrogallol autoxidation assay confirmed that the transformant strains expressed the mutated hCu, Zn-SOD protein. And the level of the mutated hCu, Zn-SOD protein reached a value of 3.61% of the total soluble protein. Furthermore, the transformants still retained 95% activities of SOD after 30 minutes at 80 degrees C environment, it indicated that the mutated hCu, Zn-SOD protein could endure higher temperature than the natural one.

Blotting, Western↗

[Study on several systems used for chemiluminescent determination of horseradish peroxidase].

Four systems were studied used for chemiluminescent determination of HRP and the determination conditions optimized. In luminol-H2O2 (NaOH) system, HRP was determined conveniently in the range of 0.05-4 pmol, with a correlation coefficient of 0.998 for the linear log-log equation and a limit of detection of 16 fmol HRP. In pyrogallol-H2O2 (pH 6.5) system, 0.1-100 pmol of HRP were determined, based on three log-log regression equations with correlation coefficients of 0.997, 0.994 and 0.999, respectively, and limits of detection of 3-17 fmol HRP. Luminol-H2O2-para iodophenol system was used to determine HRP in the range to 10-50 fmol, based on the chemiluminescence intensity at 1 min and the correlation coefficient of the log-log equation being 0.996 with a limit of detection of 10 fmol HRP. When p-hydroxybiphenyl was used to take the place of p-iodophenol and the chemiluminescence intensity at 1 min or 3 min measured, 6-40 fmol HRP were determined with a correlation coefficient of 0.994 or 0.995 for the log-log equation and a limit of detection of 6.5 or 3.3 fmol HRP.

Horseradish Peroxidase↗

[An application of electrochemical detector to dental pharmacology].

The electrochemical detection (ECD) is highly sensitive and can distinguish selectively electrochemically active materials. A sensitive and selective ECD is widely used for high-performance liquid chromatography (HPLC) of phenolic compounds, such as catecholamines, indoleamines and phenolic steroids in biological materials. In the present study, the HPLC/ECD system was applied for the simultaneous determination of various dental drugs, such as phenol, guaiacol, m-cresol, o-chlorophenol, p-chlorophenol, m-chlorophenol, eugenol, pyrogallol and resorcin. As a compromise between sensitivity and stability, the applied voltage was set at 1.2 V vs the reference electrode. These drugs were rapidly and selectively separated on a reversed-phase column using 10 mM acetate buffer (pH 4.0)-methanol (7:3, v/v) as a mobile phase. The detection limits of these drugs were determined to be 0.25 pmol per injection. These results imply that the HPLC/ECD method should be capable of determining these dental drugs with high sensitivity. The ECD was also applied to determine the enzyme activity. A highly sensitive and specific assay for alkaline and acid phosphatases and N-acetyl-beta-glucosaminidase in biological materials, such as plasma, saliva and bone, has been established. Phenol, formed enzymatically from the substrate, was determined by HPLC/ECD. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 0.5 pmol. Thus, as little as 0.5 microliters of rat plasma or 10 microliters of human saliva is all that is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in human saliva and in rat saliva elicited by pilocarpine and isoproterenol were determined. It will be possible to apply this new assay method to extremely small biological samples. Thus, the application of the HPLC/ECD system to determine dental drugs and enzyme activity with high sensitivity should prove to be of great value in the field of dental pharmacology.

Acid Phosphatase↗