Mosquito larvicidal and pupicidal activity of Euphorbia hirta Linn. (Family: Euphorbiaceae) and Bacillus sphaericus against Anopheles stephensi Liston. (Diptera: Culicidae).

OBJECTIVE: To explore the larvicidal and pupicidal activity of Euphorbia hirta (E. hirta) leaf extract and Bacillus sphaericus (B. sphaericus) against the malarial vector, Anopheles stephensi (An. stephensi). METHODS: The larvicidal and pupicidal activity was assayed against An. stephensi at various concentrations ranging from (75-375 ppm) under the laboratory as well as field conditions. The LC(50) and LC(90) value of the E. hirta leaf extract was determined by probit analysis. RESULTS: The plant extract showed larvicidal effects after 24 h of exposure; however, the highest larval mortality was found in the methanol extract of E. hirta against the first to fourth instars larvae and pupae of values LC(50)= 137.40, 172.65, 217.81, 269.37 and 332.39 ppm; B. sphaericus against the first to fourth instars larvae and pupae of values LC(50)= 44.29, 55.83, 68.51, 82.19 and 95.55 ppm, respectively. Moreover, combined treatment of values of LC(50)= 79.13, 80.42, 86.01, 93.00 and 98.12 ppm, respectively. No mortality was observed in the control. CONCLUSIONS: These results suggest methanol leaf extracts of E. hirta and B. sphaericus have potential to be used as an ideal eco-friendly approach for the control of the malarial vector, An. stephensi as target species of vector control programs. This study provides the first report on the combined mosquito larvicidal and pupicidal activity of this plant crude extract and bacterial toxin against An. stephensi mosquitoes.

Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities.

OBJECTIVE: To develop a novel approach for the green synthesis of silver nanoparticles using aqueous leaves extracts of Catharanthus roseus (C. roseus) Linn. G. Don which has been proven active against malaria parasite Plasmodium falciparum (P. falciparum). METHODS: Characterizations were determined by using ultraviolet-visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray and X-ray diffraction. RESULTS: SEM showed the formation of silver nanoparticles with an average size of 35-55 nm. X-ray diffraction analysis showed that the particles were crystalline in nature with face centred cubic structure of the bulk silver with the broad peaks at 32.4, 46.4 and 28.0. CONCLUSIONS: It can be concluded that the leaves of C. roseus can be good source for synthesis of silver nanoparticle which shows antiplasmodial activity against P. falciparum. The important outcome of the study will be the development of value added products from medicinal plants C. roseus for biomedical and nanotechnology based industries.

Mitigation of age-dependent oxidative damage to DNA in rat heart by carnitine and lipoic acid.

Endogenous damage to mtDNA by free radicals is believed to be a major contributory factor to aging. Mitochondrial DNA exists in a highly genotoxic environment created by exposure to reactive oxygen species and thus are more vulnerable to free radical attack. In the present study we have focused on the age associated alterations to DNA during aging and in parallel investigated the efficacy of carnitine (300 mg/kg bw) and lipoic acid (100 mg/kg bw) for 28 days in altering these changes. We observed a decline in the content of both mitochondrial and nuclear DNA during aging with an exponential increase in the 8-OHdG levels. We also observed an age-dependent increase in DNA protein crosslinks and double strand and single strand breaks. Supplementation of carnitine and lipoic acid during aging process decreased the incidence of these DNA damage, therefore suggesting that this feeding regimen inhibits the accumulation of age-associated oxidative DNA damage.

Supplementation of ascorbic acid and alpha-tocopherol prevents arsenic-induced protein oxidation and DNA damage induced by arsenic in rats.

Contamination of arsenic in drinking water is associated with several human diseases including cancer. It has been reported that oxidative stress plays a vital role in arsenic-induced biochemical and molecular alterations. The aim of the present study was to improve the understanding of arsenic-induced oxidative damage to proteins and to DNA and the role of antioxidants such as ascorbic acid and alpha-tocopherol in alleviating arsenic-induced damages in experimental rats. A significant increase in the levels of protein oxidation, DNA strand breaks, and DNA-protein cross-links was observed in blood, liver, and kidney of rats exposed to arsenic (100 ppm in drinking water) for 30 days. Co-administration of ascorbic acid and alpha-tocopherol to arsenic-exposed rats showed a substantial reduction in the levels of arsenic-induced oxidative products of protein and DNA. The results of this study support that free radical-mediated toxic manifestations of arsenic and also suggest that ascorbic acid and alpha-tocopherol supplementation can improve the arsenic-induced molecular alterations.

Mitochondrial membrane damage during aging process in rat heart: potential efficacy of L-carnitine and DL alpha lipoic acid.

Mitochondria are the main intracellular source of oxidizing free radicals and these oxidants produced exhibit selectivity in damaging mitochondrial macromolecules and membrane functions. In the present study we have investigated the effect of co-supplementation of carnitine (300 mg/kg bw) and lipoic acid (100 mg/kg bw) for 28 days in young, middle aged and aged rats and evaluated the effect of these compounds on age-related alterations in mitochondrial membrane functions. The levels of H2O2 were increased in both middle aged and aged rats with a concomitant decrease in the levels of cardiolipin and mitochondrial membrane potential. The levels of membrane bound ATPases were also decreased in aged rats along with alterations in mitochondrial morphology. Supplementation of carnitine and lipoic acid to middle aged and aged rats brought these changes to near normalcy. Thus, lipoic acid acts with carnitine to improve mitochondrial-supported bioenergetics and also improves general antioxidant status, thereby effectively attenuating any putative increase in oxidative stress with age.

Age-associated deficit of mitochondrial oxidative phosphorylation in skeletal muscle: role of carnitine and lipoic acid.

Mitochondrial damage has implicated a major contributor for ageing process. In the present study, we measured mitochondrial membrane swelling, mitochondrial respiration (state 3 and 4) by using oxygen electrode in skeletal muscle of young (3-4 months old) and aged rats (above 24 months old) with supplementation of L: -carnitine and DL: -alpha-lipoic acid. Our results shows that the mitochondrial membrane swelling and state 4 respiration were increased more in skeletal muscle mitochondria of aged rats than in young control rats, whereas the state 3 respiration, respiratory control ratio (RCR) and ADP:O ratio decreased more in aged rats than in young rats. After supplementation of carnitine and lipoic acid to aged rats for 30 days, the state 3 respiration and RCR were increased, whereas the state 4 and mitochondrial membrane swelling were decreased to near normal rats. From our results, we conclude that combined supplementation of carnitine and lipoic acids to aged rats increases the skeletal muscle mitochondrial respiration, thereby increasing the level of ATP.

Protective role of vitamin E on the oxidative stress in Hansen's disease (Leprosy) patients.

BACKGROUND: A constellation of reactive oxygen species (ROS) capable of damaging cellular constituents generated in excess during the chronic, inflammatory, neurodegenerative disease process of leprosy. The consequences of this leads to enhanced oxidative stress and lower antioxidant status. Enzymatic antioxidants provide first line defense against ROS. We have measured the levels of oxidative stress indices like lipid peroxidation (LPO), protein carbonyls together with enzymatic antioxidants in the blood samples of control and leprosy patients. Nutritional rehabilitation by way of exogenous supplementation of functionally efficient antioxidants like vitamin E reactivates the enzymatic antioxidant system and guards against the insult caused by ROS during the pathogenesis of the disease and antileprosy chemotherapy. DESIGN: Untreated leprosy patients were selected on the basis of clinical examination and skin smear. All diagnosed untreated leprosy patients received multi drug therapy (MDT) consisting of rifampicin, dapsone and clofazimine as recommended by World Health Organization. A small number of untreated cases were selected for co-supplementation of vitamin E along with MDT. Oxidative stress indices, enzymatic and nonenzymatic antioxidant status were assayed in untreated, MDT treated and those supplemented vitamin E along with MDT. STATISTICAL METHODS: We have compared the significance in the mean+/-s.d. values of the oxidative stress indices and the levels of antioxidants using one way analysis of variance (ANOVA) between control, untreated, MDT treated and those supplemented vitamin E with MDT and the results were significant at P < 0.05. Statistical analysis of the results suggests that oral administration of vitamin E lowers oxidative stress and augments antioxidant status in affected individuals. RESULTS: Enhanced oxidative stress as evidenced by increased LPO and protein carbonyl in leprosy cases lowers the antioxidant status. Treatment with MDT has a limited impact on increased oxidative stress and decreased antioxidant status. Coadministration of vitamin E along with MDT decreases oxidative stress and activate the antioxidant status. DISCUSSION: The excess production of ROS as seen in leprosy cases could lead to degeneration of tissues and derangement of internal organs. The possible reason for the decreased antioxidant status in leprosy cases may be increased production of ROS, deranged liver function, and the free radical producing ability of drugs used in MDT of leprosy. Intervention with antioxidant supplementation like vitamin E prevents oxidative stress mediated through ROS and activates the net antioxidant status during the chronic course of the disease and antileprosy chemotherapy.

Efficacy of levo carnitine and alpha lipoic acid in ameliorating the decline in mitochondrial enzymes during aging.

BACKGROUND: Mitochondria are central to energy production and are therefore fully integrated into the rest of the cell's physiological responses to stress. The age-related decline of capacity of each cell to manufacture energy (as ATP) is due to the progressive loss of structural integrity of mitochondria. It is apparent that as the body ages, the cells become less and less able to maintain threshold levels of cellular energy production. METHODS: In the present study we have evaluated the efficacy of carnitine, a mitochondrial metabolite and lipoic acid, a potent antioxidant on the activities of the tri carboxylic acid (TCA) cycle enzymes like succinate dehydrogenase, malate dehydrogenase, alpha-ketoglutarate dehydrogenase, Isocitrate dehydrogenase and electron transport complex I-IV in young and aged heart mitochondria. RESULT: We observed that there was an age-dependent decrement in the levels of the TCA cycle enzymes and electron transport chain complexes. Supplementation of carnitine (300 mg/kg bw/day) and lipoic acid (100 mg/kg bw/day) for 30 days brought the activities of these enzymes to almost near normal levels. CONCLUSION: These findings suggest that the combination of these drugs raises the mitochondrial energy producing capabilities by reversing the age-associated decline in mitochondrial enzyme activities and thereby protecting mitochondria from aging.

Brain regional responses in antioxidant system to alpha-lipoic acid in arsenic intoxicated rat.

Impaired antioxidant defense mechanisms and oxidative stress are implicated in the pathogenesis of arsenic toxicity. Our study was designed to determine whether alpha-lipoic acid, which has been shown to have substantial antioxidant properties, when administered (70 mg/kg body weight) once daily for 60 days along with arsenic (100 ppm sodium arsenite mixed in drinking water) would prevent arsenic-induced changes in antioxidant defense system, superoxide dismutase (SOD-total SOD, Mn SOD, Cu/Zn SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in rat brain regions such as cortex, hypothalamus, striatum, cerebellum and hippocampus. The present study also examined the effect of alpha-lipoic acid over arsenic-induced oxidant production and lipid peroxidation level (LPO) in discrete brain regions of rats. The cortex, striatum and hippocampus showed greater decreases in GSH-Px enzyme activity than cerebellum and hypothalamus with arsenic exposure. Striatum had the greatest percentage of decreased activities of total SOD and Mn SOD, whereas cortex had the greatest percentage decrease in the activity of Cu/Zn SOD in arsenic-alone treated rats. Hypothalamus and cerebellum exhibited the lowest catalase activity among all tested regions in arsenic-only treated rats. Rate of dichlorofluorescin oxidation, an indication of reactive oxygen species and other intracellular oxidants production was increased with arsenic exposure in all brain regions studied. Cortex, hippocampus and striatum exhibited greater increase of LPO levels than cerebellum and hypothalamus. SOD, CAT, GSH-Px activities were upregulated in arsenic plus lipoic acid treated versus arsenic-only treated rats. Also, simultaneous lipoic acid treatment along with arsenic proved to be sufficient in reducing oxidant production and LPO level in all rat brain regions. Our results demonstrate that arsenic-induced deficits in antioxidant enzyme activities and increase in oxidant production and lipid peroxidation level in brain regions can be overcome through simultaneous treatment with lipoic acid.

Supplementation of L-carnitine improves mitochondrial enzymes in heart and skeletal muscle of aged rats.

Aging is characterized by a general decline in physiological functions that affects many tissues and increases the risk of death. Deterioration of mitochondria, the major source and target of reactive oxygen species (ROS), is implicated in aging and a variety of age-related diseases. In the present study, the activities of citric acid cycle enzymes, such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase, were found to be decreased in aged rats as well as that of electron-transferring enzymes such as NADH dehydrogenase and cytochrome c oxidase. After supplementation of carnitine to aged rats, the activities of these enzymes reverted nearer to that of young control rats. These findings suggest that L-carnitine improves the activities of mitochondrial enzymes, increases the electron flow through the electron transport chain, and improves reducing equivalence, thereby improves energy status in aged rats.

Effect of L-carnitine on nucleic acid status of aged rat brain.

The accumulation of damage to DNA plays a significant role in the etiology of the aging process. The importance of nutrition in delaying the aging process is well recognized. L-carnitine is a quaternary ammonium compound heterogeneously distributed in the brain. In the present study the effect of L-carnitine on DNA damage of various brain regions was investigated in a duration dependent way. Male albino rats aged 4 and 24 months were administered L-carnitine (300 mg/kg body weight/day) for 7,14 and 21 days. The activities of antioxidant enzymes, the levels of nucleic acids and the extent of DNA damage were measured in cortex, hippocampus, striatum, hypothalamus and cerebellum. Our results clearly showed that the activities of super oxide dismutase, glutathione peroxidase and the levels of DNA and RNA were significantly low in cortex, hippocampus and striatum of aged rat brain when compared with that of young rats. The regions that have lower antioxidants status are highly susceptible to oxidative DNA damage. Treatment with L-carnitine in aged rats enhanced the nucleic acid, antioxidant activity in a duration dependent manner with maximal effect after 21 days whereas no significant changes could be observed in the brain of young rats. These results suggest that that L-carnitine administration prevents age-related increment of DNA damage, thereby confirming the neuroprotective action of L-carnitine against aging.

L-carnitine and DL-alpha-lipoic acid reverse the age-related deficit in glutathione redox state in skeletal muscle and heart tissues.

In the present study, the glutathione redox system was evaluated as a function of age in rat heart and muscle. A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether L-carnitine and DL-alpha-lipoic acid could compensate for GSH depletion in protection against oxidative insults. In this study we determined reduced glutathione, oxidized glutathione (GSSG), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) in skeletal muscle and heart of young and aged rats. We also calculated GSH/GSSG molar ratio and glutathione redox system. GSH levels were significantly lowered in aged rats than young rats. Conversely, GSSG levels were significantly high in aged rats. GSH/GSSG molar ratio and redox index were found to decreased in aged rats. The activities of GPx, GR, and G6PDH were found to be decreased in aged rats when compared with young rats. Supplementation of carnitine and lipoic acid to aged rats significantly increased the GSH levels thereby increasing the activity of GPx, GR, and G6PDH in skeletal muscle and heart of aged rats. In conclusion, our study suggests that supplementation of carnitine and lipoic acid to aged rats improves the glutathione redox system.

Alpha-lipoic acid increases Na+K+ATPase activity and reduces lipofuscin accumulation in discrete brain regions of aged rats.

A convincing link between oxidative stress and neurodegenerative diseases has been found with the knowledge that it actually damages neuronal cells in culture. We analyzed the effect of DL-alpha-lipoic acid on lipofuscin and Na(+)K(+) ATPase in discrete brain regions of young and aged rats. In aged rats, the level of lipofuscin was increased, and the activity of Na(+)K(+)ATPase was decreased. Intraperitoneal administration of lipoic acid to aged rats led to a duration-dependent reduction and elevation in lipofuscin and enzyme activity, respectively, in the cortex, cerebellum, striatum, hippocampus, and hypothalamus of the brain. These results suggest that lipoic acid, a natural metabolic antioxidant, should be useful as a therapeutic tool in preventing neuronal dysfunction in aged individuals.

Protective role of ascorbic acid and alpha-tocopherol on arsenic-induced microsomal dysfunctions.

Arsenic, a naturally occurring element, is present in food, soil, air and water. All human populations are exposed to arsenic and its compounds through occupational or environmental processes. Since arsenic compounds have been shown to exert their toxicity chiefly by generating reactive oxygen species, we have evaluated the effect of ascorbic acid and alpha-tocopherol on oxidative damage, antioxidant status and on xenobiotic metabolizing systems in arsenic-exposed rat liver and kidney microsomes. Arsenic exposure increases oxidative damage to lipids and proteins and decreases the levels of antioxidants and the activities of xenobiotic metabolizing enzymes. Coadministration of ascorbic acid and alpha-tocopherol to arsenic-exposed rats resulted in a reduction in the levels of lipid peroxidation, protein carbonyls and hydrogen peroxide and an elevation in the levels of reduced glutathione, ascorbic acid and alpha-tocopherol. Ascorbic acid and alpha-tocopherol treatment decreases the activity of haem oxygenase, whereas it increases the levels/ activity of cytochrome P450, cytochrome b5 and NADPH-cytochrome P450 reductase in arsenic-intoxicated rats. The results of this study provide evidence that ascorbic acid and alpha-tocopherol supplementation can improve the arsenic-induced altered microsomal functions in liver and kidney.

Effect of DL-alpha-lipoic acid on the status of lipid peroxidation and antioxidant enzymes in various brain regions of aged rats.

The effect of DL-alpha-lipoic acid on lipid peroxidation and antioxidant enzymes were evaluated in various brain regions of young and aged rats. Lipoate contents of discrete brain regions were also measured. In aged rats, the activities of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase were low whereas thiobarbituric acid reactive substances were found to be high. Catalase activity in various brain regions was little altered in aged rats. Lipoic acid an antioxidant was administered intraperitoneally (100mg/kg body weight per day) for 7 and 14 days. Lipoate administered aged rats showed a duration dependent reduction in the level of lipid peroxidation and elevation in the activities of antioxidant enzymes. There was a rise in the level of lipoate in aged rats after supplementation of lipoate in all the brain regions examined. From our results we conclude that lipoate supplementation had a beneficial effect in both preventing and reversing abnormalities in ageing brain. This beneficial effect was associated with normalization of lipid peroxidation and partial restoration in the activities of various enzymatic antioxidants suggesting that lipoate supplementation could improve brain antioxidant functions in the elderly.

Effect of L-carnitine on brain lipid peroxidation and antioxidant enzymes in old rats.

The effect of L-carnitine on lipid peroxidation and enzymatic antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase, was evaluated in brain regions of young and old rats. In all brain regions except the hypothalamus, lipid peroxidation was higher for old rats than for young control rats. The activity of superoxide dismutase, glutathione peroxidase, and catalase was lower in the striatum, cerebral cortex, and hippocampus, but no difference was observed in the hypothalamus and cerebellum. L-Carnitine administration (intraperitoneally) prevented thiobarbituric acid-reactive substance formation in the cerebral cortex, cerebellum, hypothalamus, hippocampus, and striatum of 24-month-old rats. Administration of L-carnitine reversed the age-associated changes in a duration-dependent manner. Results suggest that the neuroprotective effect on the brains in old rats was achieved by the elevation of antioxidants with L-carnitine.

Neurochemical changes related to ageing in the rat brain and the effect of DL-alpha-lipoic acid.

Age-related impairments of cognitive and motor function have been linked to a number of deleterious morphological and functional changes involving different areas of the brain. Loss of neurotransmitters, their receptors and responsiveness to neurotransmitters are key manifestations of neurological ageing and age-related disorders. In the present investigation we have evaluated the effect of DL-alpha-lipoic acid on neurotransmitters in discrete brain regions of young and aged rats. The levels of neurotransmitters were found to be lowered in aged rats. Moreover, DL-alpha-lipoic acid treated aged rats showed a increase in the status of dopamine, serotonin and norepinephrine. The results of this study provide evidence that DL-alpha-lipoic acid (a potent antioxidant) treatment can improve neurotransmitters during ageing. Hence, it can be concluded that DL-alpha-lipoic acid act as a potent neuromodulator in the brain of aged rats.

Effects of ascorbic acid and alpha-tocopherol on arsenic-induced oxidative stress.

Arsenic is an ubiquitous element in the environment causing oxidative burst in the exposed individuals leading to tissue damage. Antioxidants have long been known to reduce the free radical-mediated oxidative stress. Therefore, the present study was designed to determine whether supplementation of alpha-tocopherol (400 mg/kg body weight) and ascorbic acid (200 mg/kg body weight) to arsenic-intoxicated rats (100 ppm in drinking water) for 30 days affords protection against the oxidative stress caused by the metalloid. The arsenic-treated rats showed elevated levels of lipid peroxide, decreased levels of non-enzymatic antioxidants and activities of enzymatic antioxidants. Administration of alpha-tocopherol and ascorbic acid to arsenic-exposed rats showed a decrease in the level of lipid peroxidation (LPO) and enhanced levels of total sulfhydryls, reduced glutathione, ascorbic acid and alpha-tocopherol and so do the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase to near normal. These findings suggest that alpha-tocopherol and ascorbic acid prevent LPO and protect the antioxidant system in arsenic-intoxicated rats.

Effect of DL-alpha-lipoic acid on glutathione metabolic enzymes in aged rats.

Ageing is characterized by a failure to maintain homeostasis under conditions of physiological stress, with an increasing susceptibility to disease and death. The accumulation of errors committed by faulty biochemical reactions over a vast period generates the cumulative effect observed during ageing. The most notable among the effects of ageing are the age-related disorders where free radicals are the major cause. When the level of free radicals increases because of diet, lifestyle, environment or other influences, it results in subsequent reduction of antioxidants. Reduced glutathione is one of the most fascinating molecules virtually present in all animal cells in often quite higher concentrations. An essential mechanism that accounts for most of the metabolic and cell regulatory properties of glutathione is the thiol disulfide exchange equilibria. We evaluated the age-associated alterations in glutathione dependent enzymes, glutathione and hydroxyl radicals in young and aged rats with respect to lipoate supplementation. In aged rats, activities of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase and the level of glutathione were low, whereas the level of hydroxyl radical was higher than in the young ones. Administration of DL-alpha-lipoic acid, a thiol antioxidant intraperitoneally to the aged rats, led to a time-dependent reduction in hydroxyl radicals and elevation in the activities/level of glutathione systems. Hence it can be suggested that lipoate, a dithiol prevents the oxidation of reduced glutathione and protects its related enzymes from peroxidative damage.

Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats.

Mitochondrial dysfunction appears to contribute to some of the loss of function accompanying ageing. Mitochondria from aged tissue use oxygen inefficiently impairing ATP synthesis and results in increased oxidant production. A high flux of oxidants not only damages mitochondria, but other important cell biomolecules as well. In the present investigation, the levels of lipid peroxidation, oxidized glutathione, non-enzymatic antioxidants and the activities of mitochondrial enzymes were measured in liver and kidney mitochondria of young and aged rats before and after lipoic acid supplementation. In both liver and kidney increase in the levels of mitochondrial lipid peroxidation and oxidized glutathione and decrease in the levels of antioxidants and the activities of mitochondrial enzymes were observed in aged rats. DL-alpha-lipoic acid supplemented aged rats showed a decrease in the levels of lipid peroxidation and oxidized glutathione and increase in the levels of reduced glutathione, vitamins C and E and the activities of mitochondrial enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Thus, lipoic acid reverses the age-associated decline in endogenous low molecular weight antioxidants and mitochondrial enzymes and, therefore, may lower the increased risk of oxidative damage that occurs during ageing. From our results it can be concluded that lipoic acid supplementation enhances the activities of mitochondrial enzymes and antioxidant status and thereby protects mitochondria from ageing.