Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

Intermittent hypoxia preconditioning (IHP) has been shown to protect neurons against ischemic stroke injury. Studying how proteins respond to IHP may identify targets that can help fight stroke. The objective of the present study was to investigate whether mitochondrial dihydrolipoamide dehydrogenase (DLDH) would respond to IHP and if so, whether such a response could be linked to neuroprotection in ischemic stroke injury. To do this, we subjected male rats to IHP for 20 days and measured the content and activity of DLDH as well as the three α-keto acid dehydrogenase complexes that contain DLDH. We also measured mitochondrial electron transport chain enzyme activities. Results show that DLDH content was indeed upregulated by IHP and this upregulation did not alter the activities of the three α-keto acid dehydrogenase complexes. Results also show that the activities of the five mitochondrial complexes (I-V) were not altered either by IHP. To investigate whether IHP-induced DLDH upregulation is linked to neuroprotection against ischemic stroke injury, we subjected both DLDH deficient mouse and DLDH transgenic mouse to stroke surgery followed by measurement of brain infarction volume. Results indicate that while mouse deficient in DLDH had exacerbated brain injury after stroke, mouse overexpressing human DLDH also showed increased brain injury after stroke. Therefore, the physiological significance of IHP-induced DLDH upregulation remains to be further investigated.

Purification of procyanidins from Kunlun Chrysanthemum by macroporous resins combined with silica gel and evaluation of antioxidant activities in vitro.

The objective of the present study was to evaluate an efficient purifying process of Kunlun Chrysanthemum procyanidins (KCPC) by combination of AB-8 macroporous adsorption resin and silica gel column through adsorption and desorption experiments under static and dynamic status with the purity of procyanidins and the antioxidant activity as indexes respectively. The optimum parameters for adsorption by AB-8 resin were as follows: Sample concentration 1mg/mL, pH6, injecting velocity 2 bed volume (BV) /h, with 3 BV 70% ethanol as eluting solvent, and elution flow rate 2BV /h. One cycle after treatment using AB-8 resin, the purity of KCPC obviously enhanced 3 times compared with that of not prepurified, that is from 22.68 to 63.76%. The optimum parameters for adsorption by silica gel column were as follows: The concentration of procyanidins in a sample solution of 1.2mg/mL (pH 6) with a speed of 2BV/h. Concentration for desorption, with 5 BV of 80% ethanol as an eluent at a flow rate of 2 BV/h. After one-run treatment with silica gel column, the purity of KCPC increased from 63.76 to 81.97%. The antioxidant activities of the purified KCPC in vitro were further investigated. The results indicated that the purification method of combination of AB-8 resin and silica gel column was superior to AB-8 adsorption resin used alone in term of antioxidant activities. Moreover, the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) scavenging ability, the scavenging activity of hydroxyl radicals and the reducing ability appeared to be dose-dependent of KCPC. The novel purification method of combination of AB-8 resin and silica gel column will offer a promising way to purify KCPC for wider application.

The in vitro and in vivo antioxidant properties of Cyperus esculentus oil from Xinjiang, China.

BACKGROUND: Cyperus esculentus oil is also known as the new health care oil. This study aimed to analyse the fatty acid profile and content of C. esculentus oil by gas chromatography-mass spectrometry and to assess the antioxidant activity of C. esculentus oil. These properties were evaluated based on the reducing power, hydroxyl radical and diphenyl picryl hydrazyl radical scavenging, and a combination of in vitro and in vivo antioxidant experiments. RESULTS: Composition analyses showed that C. esculentus oil contained more than 800 g kg(-1) unsaturated fatty acid, of which oleic acid accounted for 691.4 g kg(-1), linoleic acid accounted for 107.0 g kg(-1) and palmitic acid accounted for 158.0 g kg(-1). In vitro results showed that the total antioxidant activity and the scavenging capacity of hydroxyl radicals and diphenyl picryl hydrazyl radicals increased with increasing concentration. The in vitro antioxidant ability increased as the concentration of C. esculentus oil, with 15 mL kg(-1) BW day(-1) being established as the optimal dose. CONCLUSION: Taken together, the results of this study clearly indicate that C. esculentus oil has good antioxidant properties.

The formation mechanisms of key flavor substances in stinky tofu brine based on metabolism of aromatic amino acids.

Understanding the formation mechanism of the flavor compounds in stinky tofu brine is crucial for controlling the flavor quality of Changsha stinky tofu. Dynamic changes in associated bacteria, enzymes, and differential metabolites in the metabolic pathway of aromatic amino acids in brine were investigated. Results showed that phenol (0.39～89.96 µg/mL), p-cresol (0.19～389.62 µg/mL), indole (1.14～242.97 µg/mL), 3-methylindole (0.14～3.00 µg/mL) were the key flavor substances of brine. The main associated bacteria Clostridiales bacterium SYSU GA17129, Aneurinibacillus aneurinilyticus, and Anaerosalibacter massiliensis were significantly positively correlated with key flavor substances (P < 0.05). The main associated enzymes were transaminase, decarboxylase, and lyase. In summary, phenol and p-cresol were formed by the metabolism of tyrosine and phenylalanine through five reaction chains, and indole and 3-methylindole were formed by the metabolism of tryptophan through one and three reaction chains, respectively.

Antioxidative and Hypoglycemic Effect of Ta-ermi Extracts on Streptozotocin-Induced Diabetes.

INTRODUCTION: The purpose of the present study was to reveal the potential positive effect of the Ta-ermi extracts on oxidative stress and streptozotocin (STZ)-diabetic mice and rats treated with Ta-ermi water- and alcohol-extracts. METHODS: The study was carried out using three experimental model: 1) in vitro experiments whereby Ta-ermi extracts were incubated with free radical generators such as 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to evaluate Ta-ermi's antioxidant effects; 2) testing the hypoglycemic effects of Ta-ermi extracts in streptozotocin (STZ)-induced diabetic mice; and 3) testing the beneficial effects of Ta-ermi extracts on mitochondrial complex I function using STZ-diabetic rats. RESULTS: In vitro antioxidant experiments showed that both of the extracts could scavenge free radicals and exhibited inhibitory effects on glucosidase and aldose reductase with differential effects between water extract and alcohol extract. In the STZ mouse diabetic model, both the water- and alcohol-extracts attenuated body weight decrease, decreased blood glucose levels in a concentration-dependent manner, improved insulin sensitivity, and increased oral glucose tolerance ability. In the STZ-diabetic rat model, both the water- and alcohol-extracts were found to be able to lower blood glucose levels in the diabetic animals with no effects on body weight changes. Moreover, in the STZ-diabetic rats, both the water- and alcohol-extracts of Ta-ermi could inhibit the increase of mitochondrial NADH/ubiquinone oxidoreductase (complex I) activity in the pancreas and enhanced complex I activity in the liver but showed no effect on lung or kidney mitochondrial complex I. DISCUSSION: The present study points to the potential medicinal value of Ta-ermi's water and alcohol extracts in lowering blood glucose and decreasing diabetic oxidative stress. One limitation of our study is that the compound or compounds that actually have this beneficial effect in the extracts remain unknown at this time. Therefore, the future studies should be focused on the identification of the components in the extracts that exhibit anti-oxidative and hypoglycemic effects. CONCLUSION: Taken together, our studies using different experimental paradigms indicate that Ta-ermi extracts possess antioxidant and anti-diabetic properties and may be employed as functional food ingredients for the remission of diabetes.

Neuroprotection of Cyperus esculentus L. orientin against cerebral ischemia/reperfusion induced brain injury.

Orientin is a flavonoid monomer. In recent years, its importance as a source of pharmacological active substance is growing rapidly due to its properties such as anti-myocardial ischemia, anti-apoptosis, anti-radiation, anti-tumor, and anti-aging. However, the neuroprotective effects of Orientin on stroke injury have not been comprehensively evaluated. The aim of the present study was thus to investigate the neuroprotective capacity and the potential mechanisms of Cyperus esculentus L. orientin (CLO) from Cyperus esculentus L. leaves against ischemia/reperfusion (I/R) injury using standard orientin as control. For in vitro studies, we treated HT22 cells with CoCl2 as an in vitro ischemic injury model. HT22 cells in the control group were treated with CoCl2. For in vivo studies, we used rat models of middle cerebral artery occlusion, and animals that received sham surgery were used as controls. We found that CLO protected CoCl2-induced HT22 cells against ischemia/reperfusion injury by lowering lipid peroxidation and reactive oxygen species formation as well as decreasing protein oxidation. However, CLO did not reduce the release of lactate dehydrogenase nor increase the activity of superoxide dismutase. Results showed that CLO could decrease neurological deficit score, attenuate brain water content, and reduce cerebral infarct volume, leading to neuroprotection during cerebral ischemia-reperfusion injury. Our studies indicate that CLO flavonoids can be taken as a natural antioxidant and bacteriostastic substance in food and pharmaceutical industry. The molecular mechanisms of CLO could be at least partially attributed to the antioxidant properties and subsequently inhibiting activation of casepase-3. All experimental procedures and protocols were approved on May 16, 2016 by the Experimental Animal Ethics Committee of Xinjiang Medical University of China (approval No. IACUC20160516-57).

Mitochondrial protein sulfenation during aging in the rat brain.

There is accumulating evidence that cysteine sulfenation (cys-SOH) in proteins plays an important role in cellular response to oxidative stress. The purpose of the present study was to identify mitochondrial proteins that undergo changes in cys-SOH during aging. Studies were conducted in rats when they were 5 or 30 months of age. Following blocking of free protein thiols with N-ethylmaleimide, protein sulfenic acids were reduced by arsenite to free thiol groups that were subsequently labeled with biotin-maleimide. Samples were then comparatively analyzed by two-dimensional Western blots, and proteins showing changes in sulfenation were selectively identified by mass spectrometry peptide sequencing. As a result, five proteins were identified. Proteins showing an age-related decrease in sulfenation include pyruvate carboxylase and pyruvate dehydrogenase; while those showing an age-related increase in sulfenation include aconitase, mitofilin, and tubulin (α-1). Results of the present study provide a general picture of mitochondrial protein sulfenation in brain oxidative stress and implicate the involvement of protein sulfenation in overall decline of mitochondrial function during brain aging.

Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes.

It is well established that NADH/NAD+ redox balance is heavily perturbed in diabetes, and the NADH/NAD+ redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD+ regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD+ redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and ß cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.

Hyperglycemic Stress and Carbon Stress in Diabetic Glucotoxicity.

Diabetes and its complications are caused by chronic glucotoxicity driven by persistent hyperglycemia. In this article, we review the mechanisms of diabetic glucotoxicity by focusing mainly on hyperglycemic stress and carbon stress. Mechanisms of hyperglycemic stress include reductive stress or pseudohypoxic stress caused by redox imbalance between NADH and NAD(+) driven by activation of both the polyol pathway and poly ADP ribose polymerase; the hexosamine pathway; the advanced glycation end products pathway; the protein kinase C activation pathway; and the enediol formation pathway. Mechanisms of carbon stress include excess production of acetyl-CoA that can over-acetylate a proteome and excess production of fumarate that can over-succinate a proteome; both of which can increase glucotoxicity in diabetes. For hyperglycemia stress, we also discuss the possible role of mitochondrial complex I in diabetes as this complex, in charge of NAD(+) regeneration, can make more reactive oxygen species (ROS) in the presence of excess NADH. For carbon stress, we also discuss the role of sirtuins in diabetes as they are deacetylases that can reverse protein acetylation thereby attenuating diabetic glucotoxicity and improving glucose metabolism. It is our belief that targeting some of the stress pathways discussed in this article may provide new therapeutic strategies for treatment of diabetes and its complications.

Two-dimensional gel electrophoretic detection of protein carbonyls derivatized with biotin-hydrazide.

Protein carbonyls are protein oxidation products that are often used to measure the magnitude of protein oxidative damage induced by reactive oxygen or reactive nitrogen species. Protein carbonyls have been found to be elevated during aging and in age-related diseases such as stroke, diabetes, and neurodegenerative diseases. In the present article, we provide detailed protocols for detection of mitochondrial protein carbonyls labeled with biotin-hydrazide followed by 2-dimensional isoelectric focusing (IEF)/SDS-PAGE and Western blotting probed with horse-radish peroxidase-conjugated streptavidin. The presented procedures can also be modified for detection of carbonylation of non-mitochondrial proteins.

Antioxidant and Antiproliferative Activities of Purslane Seed Oil.

The aim of this study was to evaluate the antioxidant and antiproliferative activities of PSO in vitro and its application in horse oil storage. We determined the reducing power of PSO and its scavenging effects on hydroxyl (•OH) and 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) and tested its stabilizing effects on horse oil storage. The results showed that PSO had remarkable, dose-dependent antioxidant activities, and it effectively prevented horse oil lipid oxidation. We treated cervical cancer HeLa cells, esophageal cancer Eca-109 cells and breast cancer MCF-7 cells with PSO using non-neoplastic monkey kidney Vero cells as controls. The results indicate that PSO significantly inhibited tumor cell growth in a time- and dose-dependent fashion. Our studies suggest that PSO may be used as a substitute for synthetic antioxidants in food preservation and may be potentially useful as a food and cosmetic ingredient. Meanwhile, the oxidative stress can cause hypertension, so PSO is expected to develop a health care products for the prevention and mitigation hypertensive symptoms.

Dynamic high pressure microfluidization-assisted extraction and bioactivities of Cyperus esculentus (C. esculentus L.) leaves flavonoids.

The aim of this work was to study the effect of dynamic high pressure microfluidization (DHPM) on extracting total flavonoids from Cyperus esculentus L. (C. esculentus L.) leaves and to evaluate the antioxidant activity and antibacterial property of these flavonoids. In all the assays, pretreatment with DHPM was found to not only efficiently improve the yield of total flavonoids but also strengthen the antioxidant activity of the total flavonoids. C. esculentus L. leaves flavonoids had pronounced antioxidant activity in vivo that could significantly elevate the content of superoxide dismutase (SOD) without increasing the malondialdehyde (MDA) levels, and could also improve total antioxidant capacity in mice with a dose-dependent fashion. C. esculentus L. leaves flavonoids inhibited the growth of both Gram positive and Gram negative bacteria while no obvious inhibitory effect on Penicillium and Aspergillus could be observed. Our studies indicate that flavonoids from C. esculentus L. leaves can be taken as a natural antioxidant and bacteriostatic substance in food and pharmaceutical industry.

Comparison of antioxidant and antiproliferative activity between Kunlun Chrysanthemum flowers polysaccharides (KCCP) and fraction PII separated by column chromatography.

The aim of the present study was to compare the antioxidant and antiproliferative effects on cancer cells between Kunlun Chrysanthemum flowers polysaccharides (KCCP) and its fraction PII that were separated by Biologic low pressure (LP) chromatography system followed by DEAE cellulose column chromatography. Results of in vitro experiments showed that the reducing power and the scavenging capacity of KCCP towards hydroxyl radicals (OH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals increased in a concentration dependent manner and were stronger than that of fraction PII. Results of the antiproliferative effect of KCCP and fraction PII on cervical cancer HeLa cells, esophagus cancer Eca109 cells, and mouse ascites hepatomas H22 cells indicated that both KCCP and its fraction PII possessed inhibitory activity on all the tested cancer cells at a dose- and time-dependent manner, with KCCP showing higher inhibitory activity than that of fraction PII. The present study demonstrates that KCCP and its fraction PII have antioxidant properties that may help fight cancers.

Antioxidant activity, antitumor effect, and antiaging property of proanthocyanidins extracted from Kunlun Chrysanthemum flowers.

The objective of the present study was to evaluate the antioxidant activity, antitumor effect, and antiaging property of proanthocyanidins from Kunlun Chrysanthemum flowers (PKCF) grown in Xinjiang. In vitro antioxidant experiments results showed that the total antioxidant activity and the scavenging capacity of hydroxyl radicals ((•)OH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)) radicals increased in a concentration-dependent manner and were stronger than those of vitamin C. To investigate the antioxidant activity of PKCF in vivo, we used serum, liver, and kidney from mouse for the measurement of superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (T-AOC). Results indicated that PKCF had antioxidative effect in vivo which significantly improved the activity of SOD and T-AOC and decreased MDA content. To investigate the antitumor activity of PKCF, we used H22 cells, HeLa cells, and Eca-109 cells with Vero cells as control. Inhibition ratio and IC50 values were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; PKCF showed great inhibitory activity on H22 cells and HeLa cells. We also used fruit flies as a model for analyzing the anti-aging property of PKCF. Results showed that PKCF has antiaging effect on Drosophila. Results of the present study demonstrated that PKCF could be a promising agent that may find applications in health care, medicine, and cosmetics.