[Determination of quercetin in human serum by liquid chromatography-mass spectrometry].

OBJECTIVE: To establish LC-MS method to determine quercetin in human serum. METHODS: Serum samples were extracted by acetone- methanol and hydrolyzed by ß- glucuronidase. The Agilent Poroshell 120 EC C_(18)( 50 mm × 4. 6 mm, 2. 7 µm) was used and acetonitrile( B) and 1% formic acid( A) as the mobile phase with gradient elution. Flow rate was 1. 0 m L / min and column temperature 40℃. Liquid chromatography-tandem mass spectrometry( LC-MS) with electrospray ionization( ESI~-)was performed in a negative mode and using selective negative ion detection. Detected ion m / z was 301. 1( quercetin) and 285. 1( fisetin). RESULTS: The method had good linearity in the concentration ranges of 1- 200 ng / m L. The correlation coefficient( r) was0. 9998. The detection limits of quercetin was 0. 025 ng / m L. Interday precision( RSD)was 4. 98%-8. 35%. Intraday precision( RSD) was 7. 62%-9. 73%. The recoveries of quercetin in serum was 97. 4%-112. 8%. CONCLUSION: The established method is sensitive and reproducible, and can be used in determination of quercetin in human serum.

[Effects of theanine on monoamine neurotransmitters and related genes in cerebral ischemia-reperfusion injury rats].

OBJECTIVE: To study the effects of theanine on dopamine (DA), 5-hydroxy tryptamine (5-TH) and glutamate receptor 2 (GluR2) mRNA, phospholipase-γ1 (PLC-γ1) mRNA in cerebral ischemia-reperfusion injury rats and explore the mechanism of protective effects of theanine on the induced brain injury by ischemia-reperfusion in rats. METHODS: According to random number table, a total of 56 sprague-dawley rats in SPF grade about six-week old and 100 - 120 grams weighting were divided into five groups according to the body weight levels: model group (n = 12), sham-operation group (n = 8), low theanine group (10 mg/kg), middle theanine group (30 mg/kg) and high theanine group (90 mg/kg). There were 12 rats in each of the theanine group. The rats in model group and sham-operation groups were given distilled water, and the rats in theanine groups were given corresponding theanine solution intragastrically for fifteen days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO). The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. Rats were sacrificed at 24 hours after reperfusion, the concentrations of DA, 5-HT and theanine in rats brain following ischemia-reperfusion were determined. At the same time, we determined the levels of reactive oxygen species (ROS) and activities of catalase (CAT) in mitochondria of brain. The expressions of GluR2 mRNA and PLC-γ1 mRNA in rat brain were examined by reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: The score of neurological behavior of rats in model group, theanine-low, middle, high dose groups at the 3rd hour was 6.000 ± 0.926, 4.100 ± 0.738, 3.444 ± 0.726 and 2.250 ± 0.886 respectively (F = 29.70, P < 0.01), and the score at the 24th hour in these groups was 6.625 ± 0.916, 5.000 ± 0.817, 3.667 ± 0.707 and 2.625 ± 0.916 respectively(F = 34.68, P < 0.01). The concentration of DA in model group, theanine-low, middle, high dose groups and sham-operation group was (10.26 ± 1.12), (12.48 ± 1.09), (14.55 ± 0.94), (15.97 ± 0.92) and (11.98 ± 0.63) µg/g respectively (F = 43.76, P < 0.01). The concentration of 5-HT in these groups was (1.091 ± 0.160), (0.818 ± 0.101), (0.571 ± 0.050), (0.453 ± 0.111) and (0.863 ± 0.063) µg/g respectively (F = 48.68, P < 0.01). The level of ROS was (3.072 ± 0.503), (1.331 ± 0.268), (1.295 ± 0.061), (0.804 ± 0.200) and (2.158 ± 0.218) U×min⁻¹×mg⁻¹ (F = 80.82, P < 0.01) respectively and the activities of CAT in these groups were (4.880 ± 1.121), (8.405 ± 1.356), (9.535 ± 2.511), (15.090 ± 4.054) and (21.260 ± 6.054) U/g respectively (F = 28.58, P < 0.01). The expressions of GluR2 mRNA were 0.842 ± 0.020, 1.063 ± 0.100, 1.170 ± 0.152, 1.254 ± 0.131 and 1.012 ± 0.056 respectively (F = 9.23, P < 0.01). The expressions of PLC-γ1 mRNA in these groups were 0.737 ± 0.090, 0.887 ± 0.045, 0.963 ± 0.025, 0.991 ± 0.049 and 0.867 ± 0.079 respectively(F = 10.24, P < 0.01). CONCLUSION: Theanine has a protective effect on the induced brain injury by ischemia-reperfusion in rats, which might be associated with its interaction with monoamine neurotransmitters and up-regulating the expressions of GluR2 mRNA and PLC-γ1 mRNA.

[Effects of theanine on cerebral ischemia-reperfusion injury in rats].

OBJECTIVE: To study the protective effect of theanine on cerebral ischemia-reperfusion injury induced by focal cerebral ischemia in rats. METHOD: Sprague-Dawley rats were randomly assigned into five groups: model group, shame (SH) control group and 3 theanine groups (Th-L,Th-M and Th-H). The rats in model and SH groups were given distilled water, and the rats in Th groups were given theanine solution (10, 30 and 90 mg/kg respectively) intragastrically for 15 days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO) in the model and Th groups, and SH group was used as a fake surgery control. The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. Rats were sacrificed at 24h after reperfusion, and the brain index was measured. The concentrations of aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), gamma-aminobutyricacid (GABA) and theanine (The) in rat brain following ischemia-reperfusion were determined. The expressions of BDNF mRNA and Bcl-2 mRNA in hippocampi were examined by reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Compared with model control group, the neurological deficits of theanine treated groups were milder; and the symptoms were more gently. The concentration of neurotransmitter Asp was lower while the Gly and GABA were higher, and a trend of dose-effect relation was existed. The expressions of BDNF mRNA and Bcl-2 mRNA in hippocampi were up-regulated in the theanine treated groups compared with model group (P < 0.05). CONCLUSION: Theanine has a protective effect on cerebral ischemia-reperfusion injury in rats, which may be associated with its interaction with amino acid neurotransmitters and up-regulating the expression of BDNF mRNA and Bcl-2 mRNA.

[Effect of lutein on relieving oxidative stress in mice induced by D-galatose].

OBJECTIVE: To study the effect of lutein on relieving oxidative stress in the liver of mice induced by D-galactose(D-gal). METHODS: Forty eight Kunming strain mice were randomized into 4 groups: model group, low lutein group (LL 10 mg/(kg x d)), high lutein group (HL 40 mg/(kg x d)) and normal control group. The content of reactive oxygen species (ROS), nitric oxide (NO) and activity of total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and mitochondrial Na(+)-K(+)-ATPase, Ca(2+)-ATPase in liver tissue were detected 6 weeks later in the experiment. The expression of toll-like receptor-4 (TLR4) mRNA and hemeoxygenase-1 (HO-1) mRNA in hepatic tissue were detected by reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: ROS content in HL and LL group was significantly lower (P < 0.01) than that in the model group. The activity of Na(+)- K(+)-ATPase in HL and LL group and the activity of Ca(2+)-ATPase in HL group were significantly higher than that in the model group (P < 0.05). The activities of TNOS and iNOS and the content of NO in HL group were significantly lower than the model group (P < 0.05). The expression of HO-1 mRNA in HL group was significantly higher than that in the model group, but the expression of TLR4 mRNA in HL group was significantly lower than that in the model group (P < 0.05). CONCLUSION: The mechanism of lutein on the protection of mice from oxidative stress induced by D-gal might be related to increasing the expression of HO-1 mRNA and reducing the expression of TLR4 mRNA.

[Effects of lycopene on cerebral ischemia-reperfusion injury in rats].

OBJECTIVE: To study the protective effects of lycopene (LP) on cerebral ischemia-reperfusion injury and oxidative stress in SD rats and the mechanism of them. METHODS: The rats were divided into five groups: normal control group, model control group, sham group and two LP groups (fed with 5 mg/kg bw or 20 mg/kg bw of lycopene daily for 15 days). The model for cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO). The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. The rats were put to death 24 h after reperfusion. The size of cerebral infarction was measured. The activities of iNOS, SOD, CAT and the contents of NO and MDA in brain and serum uric acid were measured. The expressions of Bcl-2 mRNA and HIF-1alphamRNA in cortex were examined by using reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: In comparison with the model group, the neurological deficits were milder, the volumes of cerebral infarction were smaller, the activities of SOD, CAT in brain tissue were higher, the activities of iNOS as well as the contents of NO, MDA in brain tissue and serum uric acid were lower in Lycopene groups. Compared with the model group and control group, the expression of HIF-1 alpha mRNA of cortex in the high dose lycopene (20 mg/kg bw) group was up-regulated; while the expression of Bcl-2 mRNA of cortex was up-regulated only in the low dose lycopene (5 mg/kg bw) group. CONCLUSION: There were some protective effects of oral administration of lycopene against cerebral ischemia-reperfusion injuries induced by focal cerebral ischemia and oxidative stress. The possible mechanism may be related with increasing activities of antioxidant enzymes, inhibiting lipid peroxidation, decreasing activities of iNOS,and up-regulating the expression of HIF-1alpha mRNA as well as Bcl-2 mRNA.

[The effects of lycopene on reactive oxygen species and anoxic damage in ischemia reperfusion injury in rats].

OBJECTIVE: To study the protective effects of lycopene (LP) on cerebral ischemia-reperfusion injury induced by focal cerebral ischemia and oxidative stress in rats. METHODS: 48 male Sprague-Dawley (SD) rats were randomly assigned into five groups: A (20 mg/kg LP), B (5 mg/kg LP), C (salad oil), D (salad oil) and E (basic feed control). A, B and C groups were given LP or salad oil orally for 15 d, then cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO) and D group was used as fake surgery control. The contents of reactive oxygen species (ROS), nitric oxide (NO), lactic acid (LD) and the activities of nitric oxide synthetase (NOS) in cortex were measured at 24 h after reperfusion. The levels of HIF-1alpha mRNA and Bcl-2 mRNA in hippocampi were determined by using reverse transcription polymerase chain reaction (RT- PCR) technique. RESULTS: ROS levels of A, B, C, D and E groups were (114.23 +/- 18.91), (135.89 +/- 14.17), (171.37 +/- 25.76), (94.24 +/- 2.23) and (92.06 +/- 5.59) fluorescence intensity value/g protein, respectively (F = 9.038, P < 0.01); levels of NO were (6.60 +/- 0.77), (7.13 +/- 0.47), (8.38 +/- 0.80), (5.52 +/- 0.16) and (5.23 +/- 0.51) micromol/g protein respectively (F = 10.197, P < 0.01); levels of NOS were (0.817 +/- 0.016), (0.875 +/- 0.095), (1.030 +/- 0.101), (0.557 +/- 0.094) and (0.595 +/- 0.066) U/mg protein respectively (F = 14.555, P < 0.01); levels of LD were (0.381 +/- 0.069), (0.446 +/- 0.012), (0.576 +/- 0.059), (0.359 +/- 0.021) and (0.310 +/- 0.036) mmol/g protein respectively (F = 10.043, P < 0.01); HIF-1alpha mRNA expression levels in hippocampi were 0.865 +/- 0.274, 0.635 +/- 0.069, 0.491 +/- 0.067, 0.375 +/- 0.052 and 0.361 +/- 0.087, respectively (F = 40.520, P < 0.01); and Bcl-2 mRNA expression levels in hippocampi were 0.263 +/- 0.033, 0.330 +/- 0.028, 0.198 +/- 0.034, 0.304 +/- 0.039 and 0.236 +/- 0.025, respectively (F = 11.003, P < 0.01). CONCLUSION: The protective effects of LP may be related with its abilities of decreasing ROS and LD cumulation, alleviating inflammation and up-regulating the expression of protective genes.

[Mechanism of protective effects of procyanidins on liver injury induced by alcohol in mice].

OBJECTIVE: To study the mechanism of protective effects of procyanidins on liver injury model induced by alcohol in mice. METHODS: Mice were divided into 3 groups to set up the model of liver injury induced by alcohol in mice: the group of liver injury induced by alcohol, the group of procyanidins with low doses (100mg/kg), the group of procyanidins with high doses(300mg/kg). Then established normal group of control. The model of liver injury induced by alcohol in mice was used to investigate the protective effects at low and high doses procyanidins in serum indicated by alanine aminotransferase (ALT), asparate aminotransferase (AST) and in liver homogenate indicated by malonaldehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS). The levels of Cu,Zn-SOD mRNA in hepatic tissue and toll like receptor 4 (TLR4) mRNA were determined by using reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: The procyanidins with low and high doses could reduce ALT, MDA, ROS content and increase SOD level, up-regulate the expression of Cu, Zn-SOD mRNA and down-regulate the expression of TLR4 mRNA. CONCLUSION: Mechanism of the protective effects on liver injuries of mice induced by alcoholmay be associated with promoting the expression of Cu, Zn-SOD and suppressing the expression of TLR4 mRNA.

[Effects of grape seed proanthocyanidins extracts on experimental diabetic nephropathy in rats].

OBJECTIVE: To study the effects of grape seed proanthocyanidins extracts(GSPE) and its mechanism on early renal lesions of diabetic rats. METHODS: Diabetic rats induced by alloxan were given GSPE intragastrically for 6 weeks, then the antioxidative indexes and NO content, NOS activity in kidney and serum were measured, and the renal function indexes were tested as well. RESULTS: Compared with the diabetic group, the urinary protein/24h, levels of blood urea nitrogen(BUN)and serum creatinine(SCr), creatinine clearance rate(CCr) and the ratio of kidney weight/body weight were decreased, the SOD activity in kidney was raised while MDA content was fall in the GSPE group(high dose), and the differences were all significant. The NO content in the kidney and NOS activity in kidney and serum decreased in the GSPE (low dose)group, and there was significant difference when compared with diabetic group( P <0.05) . CONCLUSION: GSPE has the effect in protecting kidney of diabetic rats, the mechanism might be related with its action in increasing the renal antioxidative ability,decreasing the content of NO and the activity of NOS in kidney and serum.

[Green tea extracts protected against carbon tetrachloride-induced chronic liver damage and cirrhosis].

OBJECTIVE: Using the carbon tetrachloride liver cirrhosis rat model, the protective effect of the green tea extractive (GTE) on the liver cirrhosis was studied. METHODS: Male SD rats were randomly divided into three groups: normal group, GTE group and cirrhosis group. The GTE group and the cirrhosis group were injected subcutanuously 2 times/wk over 9 weeks with 40% CCl(4). In the second and the ninth week, the rats were sacrificed to measure MDA and hydroxyproline concentrations and TGF-beta(1) mRNA expression in liver tissue, as well as to conduct histological examination on various organs. RESULTS: Compared with the cirrhosis group, the MDA and the hydroxyproline concentrations in the GTE group were significantly reduced (P < 0.05). The liver necrosis and cirrhosis were extenuated in the GTE group by means of histologic examination. The expression of the TGF-beta(1) mRNA was reduced significantly in the GTE group. CONCLUSION: Dietary supplementation of GTE can protect against CCl(4)-induced liver damage and cirrhosis in rats.