HPLC-UV method for the simultaneous determinations of ascorbic acid and dehydroascorbic acid in human plasma

A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 µg/mL. The inter- and intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.

Dehydroascorbic Acid Attenuates Ischemic Brain Edema and Neurotoxicity in Cerebral Ischemia: An in vivo Study

Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

Study on effects and mechanism of traditional Mongolian medicine wuweifengshi capsule on adjuvant arthritis in rats / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA).</p><p><b>METHOD</b>Wister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated.</p><p><b>RESULT</b>Wuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity.</p><p><b>CONCLUSION</b>The anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.</p>

Vitamina C, carotenoides, fenólicos totais e atividade antioxidante de goiaba, manga e mamão procedentes da ceasa do estado de Minas Gerais / Vitamin C, carotenoids, phenolic compounds and antioxidant activity of guava, mango and papaya from ceasa of Minas Gerais state

Este estudo teve como objetivo analisar o conteúdo de compostos antioxidantes (ácido ascórbico - AA, ácido desidroascórbico - ADA, vitamina C total, licopeno, β-caroteno, β-criptoxantina e estimativa de compostos fenólicos) e avaliar a atividade antioxidante, em goiaba, manga e mamão. A análise de carotenoides e vitamina C foi realizada por Cromatografia Líquida de Alta Eficiência (CLAE). O teor de fenólicos totais foi determinado utilizando o reagente de Folin-Ciocalteu e leitura espectrofotométrica. A atividade antioxidante foi avaliada pelo Teste do 2,2-diphenil-2-picril-hidrazil (DPPH•) e do Poder Redutor. A Anova (α= 0,05) foi utilizada para a análise dos dados. Os teores dos constituintes antioxidantes diferiram entre as três frutas, mas a goiaba foi a fruta que apresentou teores mais elevados de compostos fenólicos, vitamina C total, ADA e licopeno, além dos maiores valores para atividade antioxidante. Foi constatada forte correlação entre os testes que avaliaram a atividade antioxidante e o teor de fenólicos totais, demonstrando serem estes os principais compostos antioxidantes a contribuírem para a atividade antioxidante das frutas analisadas, em ambos os testes. É importante incentivar a utilização das frutas avaliadas neste estudo, tanto em nível doméstico quanto em estabelecimentos de alimentação coletiva para aumentar o consumo de antioxidantes naturais pela população.

This study aimed to analyze the content of antioxidant compounds (ascorbic acid - AA, dehydroascorbic acid - DHA, total vitamin C, lycopene, β-carotene, β-cryptoxanthin and phenolic compounds) and to evaluate the antioxidant activity in guava, mango and papaya. The analysis of carotenoids and vitamin C was performed by high performance liquid chromatography (HPLC). The content of phenolic compounds was determined using the Folin-Ciocalteu reagent and spectrophotometric reading. Antioxidant activity was evaluated by testing the 2.2-diphenyl-2-picryl-hydrazil (DPPH•) and reducing power. ANOVA was used for data analysis (α= 0.05). The levels of antioxidant constituents differed among the three fruits; guava was the fruit that had the highest levels of phenolic compounds, total vitamin C, lycopene and DHA, and the highest values for antioxidant activity. There was a strong correlation between tests that evaluated antioxidant activity and the content of phenolic compounds, demonstrating that these are the main antioxidant compounds to contribute to antioxidant activity of fruits examined in both tests. It is important to encourage the use of fruits evaluated in this study, both at home and in food service establishments, in order to increase the intake of natural antioxidants by the population.

Teores de B-caroteno e vitamina C durante o amadurecimento da manga "UBÀ"(Mangifera indica L. var. Ubá)

A manga Ubá é uma fruta de custo reduzido, amplamente cultivada na Zona da Mata Mineira e muito apreciada pelos consumidores. Com o objetivo de avaliar o conteúdo de antioxidantes naturais ao longo do amadurecimento da fruta, este trabalho quantificou o conteúdo de β-caroteno e vitamina C total (ácido ascórbico-AA e ácido desidroascórbico-ADA) na fruta in natura em três estádios de maturação: verde, parcialmente madura e madura. As análises foram feitas por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo médio de β-caroteno foi maior na manga madura, seguido pela manga parcialmente madura e manga verde. Para AA e vitamina C total, ao contrário, observou-se maior conteúdo na manga verde, seguido pela fruta parcialmente madura e menor conteúdo na manga verde. Para ADA não foi observada diferença entre os estádios de maturação. A manga Ubá apresentou-se como uma excelente fonte de antioxidantes naturais, com teores de β-caroteno e vitamina C superiores aos comumente encontrados em outras variedades de manga, o que pode contribuir para atender boa parte das necessidades diárias de vitaminas A e C.

Comparative research of different Bupleurum chinense composition to influence of hepatotoxicity of rats and oxidative damage mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the influence of different Bupleurun chinense composition to the degree of hepatotoxicity damage to rats and oxidative damage mechanism.</p><p><b>METHOD</b>To successively lavage alcohol extracted and water extracted B. chinense composition to rats for 30 days, the general conditions were observed and the related index of liver function, the content of total-SH in serum, the content of MDA, the activity of SOD and the content and activity of GSH and GSH-Px in serum and liver tissue were detected.</p><p><b>RESULT</b>Alcohol and water extracted B. chinense composition all could induce the increases of the activity of ALT and AST in serum, liver weight and the ratio of liver to body, and the content of MDA and induce the decreses of the content of total -SH in serum, the content of GSH, and the activity of SOD and GSH-Px in serum and liver tissue. The above-mentioned changes gradually aggravated with dose increasing, and there was significant difference compared with control group with distilled water.</p><p><b>CONCLUSION</b>The different B. chinense composition all can induce hepatotoxicity damage, and the channel of hepatic damage is related with the peroxidative damage mechanism. The degree of hepatotoxicity damage caused by the alcohol extracted composition is more serious than that by the water extracted composition.</p>

Effects of Ascorbic and Dehydroascorbic Acids on Apoptotic Cell Death in Hippocampal Slice Culture

Ascorbic acid (AA) and dehydroascorbic acid (DHA) are known to have protective effects in experimental central nerve system disorder models such as stroke, ischemia, and epileptic seizures. The present study was conducted to examine the protective effect of AA and DHA on kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures (OHSC). Protective effects of AA and DHA on KA-induced cell death were evaluated by analyzing caspase-3. In addition, to determine if the prooxidant effect of AA is related to iron, the effect of AA on cell death was examined using desferrioxamine (DFO), an iron chelator. After 12h-KA treatment, significant delayed neuronal death was detected in CA3 region, but not in CA1. The AA (500 micrometer) and DHA (100 and 500 micrometer) pretreatments significantly prevented cell death by inhibiting caspase-3 activation in CA3 region. In the concentration of 1,000 micrometer, however, AA pretreatment might have prooxidant effect, but AA-induced oxidative reaction is mainly not related to transition metal ions. These data showed that the pretreatments of intermediate-dose AA and DHA protected KA-induced neuronal damage in OHSCs and co-pretreatment of AA and DFO did not affect cell death except for a few cases. These data suggest that both AA and DHA pretreatment have antioxidant or prooxidant effect depending on doses treated on KA-induced neuronal injury and the possible prooxidant effect of AA may not depend on the Fenton reaction.

Effect of zinc on antioxidant response in maize (Zea mays L.) leaves.

Maize (Zea mays L. cv kanaujia) plants grown with Zn [10 (control), 0.1 (low) and 20 microM (high)], were investigated for concentration of antioxidants and activities of antioxidative enzymes in leaves. Young leaves of low Zn plants developed whitish-necrotic spots. Leaves of both low and high Zn plants showed decrease in chlorophyll concentration and accumulation of lipid peroxides, ascorbate and dehydroascorbate, associated with a decrease in the activity of ascorbate peroxidase and superoxide dismutase. Low and high Zn, however, showed diverse effect on glutathione reductase. While low Zn increased the activity of glutathione reductase, high Zn decreased its activity. Zinc effect on antioxidative constituents suggested Zn involvement in sustaining the antioxidative defense system in maize leaves.

Effects of glutamate on dehydroascorbate uptake and Its enhanced vulnerability to the peroxidation in cerebral cortical slices

Pro-oxidant properties of ascorbate have been studied with uses of brain tissues and neuronal cells. Here we address potential mechanism of ascorbate coupling with glutamate to generate oxidative stress, and the role which oxidized ascorbate (dehydroascorbate) transport plays in oxidative neuronal injury. Ascorbate in neurones can be depleted by adding glutamate in culture medium since endogenous ascorbate can be exchanged with glutamate, which enhances ascorbate/ dehydroascorbate transport by depleting ascorbate in the neurons with the glutamate-heteroexchange. However, ascorbate is known readily being oxidized to dehydroascorbate in the medium. Glutamate enhanced the dehydroascorbate uptake by cells via a glucose transporter (GLUT) from extracellular region, and cytosolic dehydroascorbate enhanced lipid peroxide production and reduced glutathione (GSH) concentrations. Iso-ascorbate, the epimer of ascorbate was ineffective in generating the oxidative stress. These observations support the current concept that the high rates of dehydroascorbate transport via a GLUT after the release of ascorbate by glutamate leads to peroxidation, the role of glutamate on ascorbate/ dehydroascorbate recycling being critical to induce neuronal death via an oxidative stress in the brain injury.

Contenido de vitaminas en frutillas / Content of vitamins in strawberries

Intenta determinar el contenido de vitamina C y de sus formas activas en las frutillas consumidas habitualmente en nuestro país, ya que no existen datos locales acerca del contenido de este nutriente, que en frutos presenta una gran variabilidad. Los resultados del análisis, realizado en muestras obtenidas en el Mercado Central, indican que el tiempo y las condiciones de almacenamiento afectan el contenido de vitamina

Ascorbic acid status in uremics.

Plasma levels of ascorbic acid (AA) and dehydroascorbic acid (DHA) were estimated in 27 patients of end stage renal failure (ESRF) on standard conservative therapy (group A) and 9 patients of ESRF on maintenance haemodialysis (MHD; group B). Fourteen healthy subjects matched for age and sex served as control (group C). The dietary intake of vitamin C was significantly decreased in group A than in group B compared to control. Similarly, plasma AA was significantly lowered to 0.801 +/- 0.283 mg per cent in group A compared to 1.421 +/- 0.47 mg per cent in control. While it was just lowered to 1.058 +/- 0.272 mg per cent in group B. Although plasma level of DHA was raised to 0.243 +/- 0.486 mg per cent and 0.166 +/- 0.54 mg per cent in groups A and B respectively, the increase was not statistically significant. In our present study, the DHA/AA ratio was found to be inversely proportional to the plasma AA. Further, this ratio has been claimed to be a better indicator of overall reducing atmosphere (i.e., profile of vitamin C) of the body.

Plasma dehydroascorbic acid levels in Iranian subjects with diabetes mellitus

Several reports concerning high plasma dehydroascorbic acid [DHAsA] levels in diabetics have been published and from these reports, suggestions that monitoring of DHAsA levels in those persons with a predisposition to diabetes mellitus would be of value. However, conflicting reports have also appeared which do not confirm high levels of DHAsA in diabetic subjects when compared to controls. Because of these conflicting results, this investigation using Iranian diabetic subjects was undertaken to ascertain whether or not periodic monitoring of DHAsA levels would be of value as an indicator of prediabetic conditions. Our results do not confirm high levels of DHAsA in diabetics but because of the many theories concerning the mechanism of action and the metabolism of DHAsA, previous findings cannot be disregarded

Ascorbic acid, dehydroascorbic acid and glutathione in liver disease.

Controlled studies were conducted to find out the plasma values of ascorbic acid, dehydroascorbic acid (DHA), urinary excretion of ascorbic acid and blood levels of glutathione in patients with viral hepatitis, alcoholic hepatitis, cirrhosis of liver and carcinoma of liver. Leucocyte ascorbic acid and DHA/AA index were also determined in order to assess the ascorbic acid status of these patients. It was observed that the plasma and leucocytes contents of ascorbic acid were significantly subnormal with markedly decreased urinary excretion in these patients. Decreased level of glutathione and significantly higher level of DHA reflect an over all reducing status of the body is markedly deranged in these conditions. Further it was observed that the DHA/AA ratios were significantly altered in these groups of patients.

Changes in ascorbic acid content and acetylcholinesterase activity in the muscle of frog following sciatectomy.

Unilateral-sciatectomy for three months in the frog, Rana cyanophlictis resulted in a substantial increase on unit weight basis in the ascorbic acid (ASA) and dehydroascorbic acid (DHA) contents of the sciatectomized gastrocnemius muscle. Diketogulonic acid (DKA) levels did not vary. On whole muscle-weight basis only the ASA level increased. The AChE activity in sciatectomized muscle is significantly lower than that of the control. Partially purified preparation of the AChE from the sciatectomized muscle showed different kinetics compared to that from innervated control. In vitro additions of ASA in physiological concentration to the enzyme assay medium inhibited the AChE activity significantly and the inhibition was an un-competitive type. Reduced activity of the enzyme has been correlated to the increased concentration of ASA in the sciatectomized muscle.

In vitro effect of certain compounds on the lysosomal release of polymorphonuclear leukocytes.

From studies on the release of acid phosphatase and B-glucuronidase, it was observed that quercetin, a vitamin P like compound and ascorbic acid stablise while dehydroascorbate, acetoacetate and B-hydroxybutyrate labilise the leukocytic lysosomes in vitro. These effects were compared with chloroquine and progesterone, known stabiliser and destabiliser, respectively. The possible mode of lysosomal labilisation by ketone bodies and dehydroascorbate has been suggested.