International Journal of Nutrition, Pharmacology, Neurological Diseases

: 2012  |  Volume : 2  |  Issue : 3  |  Page : 266--271

Chronotherapeutic effect of morin in experimental chronic hyperammonemic rats

Selvaraju Subash, Perumal Subramanian 
 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India

Correspondence Address:
Perumal Subramanian
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamil Nadu


Aim: Ammonia is a neurotoxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy and a major pathogenic factor associated with inborn errors of urea cycle. In this present work we aimed to evaluate the chronotherapeutic effect of morin (3,5,7,2«SQ»,4«SQ»-pentahydroxyflavone), a plant component, on ammonium chloride (AC) (100 mg/kg; intraperitoneal)-induced hyperammonemia in Wistar rats (180-200 g). Materials, Methods and Results: Morin (30 mg/kg body weight) was administered to rats at 06:00, 12:00, 18:00, and 24:00 hours in hyperammonemia. The influence of morin on AC-induced hyperammonemia at different time points (06:00, 12:00, 18:00, and 24:00 hours) was evaluated by analyzing the circulatory levels of ammonia; urea; thiobarbituric acid reactive substances (TBARS); hydroperoxides (HP); liver markers [alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP)]; glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT); reduced glutathione (GSH); and vitamins A, C, and E. The levels of these components were significantly elevated in AC-treated rats but were decreased significantly after treatment with morin. Administration of morin at 24:00 h caused significantly greater reduction in these parameters than administration at other time points (P<0.05; Duncan«SQ»s multiple range test). Conclusion: The chronotherapeutic effect of morin in hyperammonemic rats may be due to various factors, including (i) temporal variations of metabolic enzymes involved in the degradation of morin; (ii) temporal variations of lipid peroxidation and of antioxidants, urea cycle enzymes etc.; and (iii) temporal variation in bioavailability of morin. However, the exact underlying mechanism(s) is/are still unclear and further investigations are needed.

How to cite this article:
Subash S, Subramanian P. Chronotherapeutic effect of morin in experimental chronic hyperammonemic rats.Int J Nutr Pharmacol Neurol Dis 2012;2:266-271

How to cite this URL:
Subash S, Subramanian P. Chronotherapeutic effect of morin in experimental chronic hyperammonemic rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2021 Feb 25 ];2:266-271
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Hyperammonemia is defined as elevated ammonia concentration in blood, resulting from inadequate ammonia detoxification due to impairment in liver function. In living organisms, ammonia is an important nitrogen substrate in several reactions and plays an important role in nitrogen homeostasis of cells. Moreover, ammonia is a product as well as precursor of various important nitrogen-containing metabolites such as amino acids, the smallest subunits of proteins. [1] Ammonia is neurotoxic when it accumulates in excess. Hyperammonemia is mainly responsible for the neurological alterations-including impaired intellectual function-found in the hepatic encephalopathy seen in patients with liver disease. Antiepileptic drugs such as valproate and salicylate also cause hyperammonemia and urea cycle disorders (UCDs). Ammonia toxicity results in free radical generation that leads to oxidative stress-mediated tissue damage, [2],[3],[4] and elevated ammonia concentration in the brain exerts toxic effects on neural cells. [5]

Chronotherapy is a new strategy of drugs administration based on circadian rhythms. [6] The hypothalamic biological clock (suprachiasmatic nuclei) modulates the rhythms of cellular metabolism and proliferation in normal tissues. [7] Normal cell physiology is characterized by predictable changes over a 24-hour timescale, which is coordinated by the suprachiasmatic nucleus. These circadian rhythms can be utilized to reduce cytotoxic treatment insults by making temporal adjustments to the administration schedule of drugs (chronotherapy). [8] Drugs that target the regulation of cell-cycle events or angiogenesis are examples of drugs that are more effective if administered at specific times. With chronotherapy, such drugs may fully achieve in vivo the pharmacodynamic effects they display in vitro.

Preclinical and clinical evidence support investigations of the chronotherapy hypothesis in cancer patients. The recognition of the importance of diurnal patterns led to the development of the innovative technique of chronotherapy, which is the delivery of medications in synchrony with endogenous biological rhythms of the pathophysiology of disease states, so as to optimize treatment outcomes. [9] Recently, this strategy of chronotherapy has been successfully implemented in the therapy of allergic rhinitis and asthma, [9] hypertension and ischemic heart disease, [10] and cancer. [11]

The greatest disadvantage of the presently available potent conventional or synthetic antihyperammonemic agents/therapies lies in their toxicity and the tendency for reappearance of symptoms after discontinuation of treatment. These drugs or therapies are sometimes inadequately effective and have serious adverse effects. [12] Therefore screening and development of drugs for antihyperammonemic activity is still in progress and there is a worldwide trend to go back to traditional medicinal plants and natural products. There is a need to find effective natural protective agents against hyperammonemia. This can be achieved by research focusing on the active principles of plants, and there are many indications that natural products may be better than currently used drugs. Polyphenols/flavonoids occur ubiquitously in foods of plant origin [13] and have received much attention because of their broad-spectrum pharmacological activities and extensive biological effects. [14],[15] The basic structure of flavonoids is usually characterized by two aromatic rings, ring A and ring B, joined by a three-carbon-linked c-pyrone ring (ring C), forming a C 6 -C 3 -C 6 skeleton unity where polar groups-usually hydroxyl, methoxyl, or glycosyl-are appended at various positions. [16] Flavonols vary from one another on the basis of the number and position of hydroxyl groups and their pharmacological actions. Each flavonol has its own specific biological function and activity. [17]

Morin (3,5,7,2',4'-pentahydroxyflavone) is a kind of flavonoid belonging to the group of flavonols found in old fustic (Chlorophora tinctoria), osage orange (Maclura pomifera), [18] almonds (P. guajava L.), [19] mill (Prunus dulcis), fig (Chlorophora tinctoria), [20] onion and apple, [21] and other Moraceae, which are all part of the normal diet and/or are taken as herbal medicine. [22] Morin has been shown to have potent antioxidant and metal ion-chelating capacities; it has various biological and biochemical effects, including antioxidative, anti-mutagenesis, anti-inflammatory, [23],[24],[25] antineoplastic, cardioprotective, [26],[27] and anticancer [28] activities; and it is also an xanthine oxidase inhibitor, [29] protein kinase C inhibitor, [30] and cell proliferation inhibitor. [31] In addition to inhibiting P-gp, morin can modulate the activities of the metabolic enzymes, including cytochrome P450 (CYPs). [32] Furthermore, morin exerts an antitumor effect by significantly inhibiting the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen activation and TPA-induced skin tumor promotion. [33],[34] Morin has also been shown to act as a chemopreventive agent against oral carcinogenesis in vitro and in vivo.[28],[35]

Although various traditional medicinal values have been attributed to morin, no biochemical studies have been carried out to shed light on the chronotherapeutic effect of morin on circulatory liver markers and redox status in experimental hyperammonemia. Therefore, the present study was designed to analyze the chronotherapeutic effect of morin on circulatory (i) ammonia, urea, alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP); (ii) thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP); and (iii) enzymatic and nonenzymatic antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and vitamins A, C, and E in hyperammonemic rats.

 Materials and Methods

Experimental animals

Adult male albino Wistar rats (180-200 g), bred in the Central Animal House of Rajah Muthiah Medical College, Annamalai University, were used in this study. The animals were housed in polycarbonate cages in a room with a 12-hour/12-hour day-night cycle, in temperatures of 22±2°C and humidity of 45%-64%. The animals were fed with a standard pellet diet (Hindustan Lever Ltd, Mumbai, India) and provided water ad libitum. Studies were carried out in accordance with Indian national laws on animal care and use, and ethical clearance was obtained from the Committee for the Purpose of Control and Supervision of Experiments on Animals of Rajah Muthiah Medical College and Hospital (Reg. No: 160/1999/CPCSEA), Annamalai University, Annamalainagar.

Drugs and chemicals

Morin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ammonium chloride was purchased from Sisco Research Laboratories, Mumbai, India. All other chemicals used in the study were of analytical grade.

Preparation of morin

Morin was freshly dissolved in a small amount of ethanol and then diluted with physiological saline. [36]

Induction of experimental hyperammonemia

Hyperammonemia was induced in Wistar rats by intraperitoneal (ip) injections of ammonium chloride at a dose of 100 mg/kg body weight, thrice a week, for 8 consecutive weeks. [37]

Experimental design

A total of 32 rats were used in the experiment. The rats were divided into four groups of eight rats each. Group I rats received physiological saline and formed the control group; group II rats were administered morin (30 mg/kg body weight) using an intragastric tube; [38] group III rats were treated with ammonium chloride (AC; 100 mg/kg body weight, ip); [37] and groups IV-VII (at 06:00, 12:00, 18:00, and 24:00) rats treated with AC (100 mg/kg) + morin (30 mg/kg) thrice in a week for 8 weeks. At the end of 8 weeks the rats were fasted overnight and sacrificed by cervical dislocation after anesthesia with intramuscular injection of ketamine hydrochloride (30 mg/kg body weight).

Biochemical analysis

After the experimental period, blood samples were collected from the animals for biochemical estimations (groups I-IV) at (06:00, 12:00, 18:00 and 24:00). Ammonia [39] , urea [40] , AST and ALT, [41] ALP, [42] TBARS, [43] HP, [44] SOD, [45] CAT, [46] GPx, [47] vitamin A, [48] α-tocopherol,[49] vitamin C, [50] and GSH [51] levels were estimated.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) using SPSS® software v 9.05. Results were expressed as the mean value (±SD) of the eight rats in each group. P<.05 was considered as significant.


[Table 1] shows the levels of blood ammonia, plasma urea, HP, TBARS, and serum AST, ALT, and ALP in control and experimental rats. The levels of circulatory ammonia, urea, liver markers, HP, and TBARS were significantly higher in AC-treated rats when compared with control. Hyperammonemic rats treated with morin at 24:00 hours showed significant normalization of the levels of ammonia, urea, liver markers, and lipid peroxidation products as compared with control rats.{Table 1}

The levels of circulatory antioxidants in control and experimental groups are given in [Table 2]. The levels of vitamins A, C, and E; GSH; GPx; SOD; and CAT were significantly lower in AC-treated rats and these levels were significantly normalized in hyperammonemic rats treated with morin at 24:00 hours.{Table 2}


Ammonia is present in all living organisms as a product of degradation of proteins and other nitrogenous compounds. However, at high levels, ammonia is toxic and can cause functional disturbances in the central nervous system (CNS) that could lead to coma and death. To avoid the deleterious effects of ammonia, ureotelic animals detoxify ammonia by incorporating it into urea, which is then eliminated in urine. However, when the liver fails or when blood is shunted past the liver, blood ammonia levels are elevated and brain function deteriorates. [52] In the liver, ammonia is removed either in the form of urea in periportal hepatocytes and/or as glutamine in perivenous hepatocytes. [52] Increased levels of circulatory ammonia and urea indicates a hyperammonemic condition in rats treated with AC, [37] which may be due to liver damage caused by ammonia intoxication. Administration of morin at 24:00 hours to AC-induced hyperammonemic rats significantly decreased the levels of blood ammonia and urea as compared with administration of morin at other time points. The reduction in the levels of ammonia and urea during morin treatment shows the potent anti-hyperammonemic effect of morin. [38],[53],[54]

In our study, the elevated levels of circulatory liver markers and lipid peroxidation products in AC-treated rats might be due to the liver damage caused by ammonia-induced free radical generation. Reports have shown that excess ammonia intoxication leads to excessive activation of NMDA receptors, leading to neuronal degeneration and death. [55],[56] The mechanisms by which excessive activation of NMDA receptors leads to neuronal degeneration and death include increased Ca 2+ concentration in the postsynaptic neuron. [57],[58] Ca 2+ binds to calmodulin and activates nitric oxide synthase, increasing the formation of nitric oxide (NO), which contributes to the neurotoxic process. Activation of NMDA receptors also leads to increased production of superoxide radical, which has also been proposed to play a role under in vivo conditions. [59],[60] Superoxide and NO have the ability to generate hydroxyl radicals. [61] This leads to oxidative stress, which causes tissue damage. [62] Decreased levels of circulatory liver markers and lipid peroxidation products in morin-administered rats may be due to its free radical scavenging property. [63],[64] Previous studies show that morin offers neuroprotection by inhibiting excess activation of NMDA receptors and NMDA receptor-mediated neurotoxicity. The potent neuroprotective activity of morin could be of value in the treatment of acute neuronal damage and adisability. [65]

Evidence shows that the oxidative stress and free radical production could be involved in the mechanism of ammonia intoxication, [66] which might have been responsible for the decrease in the levels of enzymatic (GPx, SOD, and CAT) and nonenzymatic (GSH and vitamins A, C, and E) antioxidants in AC-treated rats. Administration of morin significantly restored the levels of enzymatic and nonenzymatic antioxidants; this may be due to its potent antioxidant property by offering possible role in reducing the oxidative stress by inducing cellular antioxidant enzymes. [64] It has been reported that flavonoids such as morin, quercetin, and kaempferol are potent free radical scavengers and antioxidants. [67] For instance, phenolic phytochemicals, due to their phenolic ring and hydroxyl substituents, can function as effective antioxidants by virtue of their ability to quench free radicals. It is therefore believed that dietary phenolic antioxidants can scavenge harmful free radicals and thus inhibit their oxidative reactions with vital biological molecules, [68] thereby preventing the development of many pathophysiological conditions. The possible mechanisms by which morin modulates liver marker levels and redox status during experimental hyperammonemia might be via removal of excess ammonia, inhibition of NMDA receptor-mediated neurotoxicity, and antioxidant activity. [69],[70]

Chronobiological studies provide the capability of therapeutic intervention at a time when this intervention is useful and best tolerated and avoidance when it is not. [71] The chronobiologic approach to treatment, by exploring the rhythmic nature of oxidants and antioxidants, is especially critical and meaningful when potentially damaging or toxic agents have to be used. But beyond this, the time factor has to be introduced in just about all aspects of clinical pharmacology and many time-honored customs like 'three times a day' medications will have to be replaced by more meaningful, and often more effective and less toxic, chronobiologic treatment schedules. [72] The choice of the 'right time' will require chronobiologic knowledge, interpretation and experience since treatment at the 'wrong time' can be potentially harmful. [71],[73]


The chronotherapeutic effect of morin (administered at 24:00 hours) in hyperammonemic rats may be due to various factors, including (i) significant variations in the pharmacokinetics of morin over the 24-hour day ; (ii) temporal variations of metabolic enzymes involved in the degradation of morin; (iii) temporal variations of liver marker enzymes, lipid peroxidation products, antioxidants, urea cycle enzymes, etc.; and (iv) temporal variation in the bioavailability of morin. However, to elucidate the underlying mechanism(s) further investigations are desirable.


Financial assistance from the University Grants Commission, New Delhi, is gratefully acknowledged.


1Shokati T. Metabolic trafficking between astrocytes and neurons under hyperammonemia and manganism: Nitrogen and Carbon metabolism. PhD thesis, Dem Fachbereich Biologie/Chemie der, Universität Bremen, vorgelegt von, 2005, 25-28.
2Vijayakumar N, Subramanian P. Neuroprotective effect of semecarpus anacardium against hyperammonemia in rats. J Pharm Res 2010;3:1564-8.
3Thenmozhi J, Subramanian P. Hepatoprotective effect of Momordica charantia in ammonium chloride induced hyperammonemic rats. J Pharm Res 2011;4:700-2.
4Zielinska M, Ruszkiewicz J, Hilgier W, Fresko I, Albrecht J. Hyperammonemia increases the expression and activity of the glutamine/arginine transporter y + LAT2 in rat cerebral cortex: Implications for the nitricoxide/cGMP pathway. Neurochem Int 2011;58:190-5.
5Kosenko E, Kaminsky Y, Stavroskaya IG, Felipo V. Alteration of mitochondrial calcium homeostasis by ammonia-reduced activation of NMDA receptors in rat brain in vivo. Brain Res 2000;880:139-46.
6Focan C. Marker rhythms for cancer chronotherapy. From laboratory animals to human beings. In Vivo 1995;9:283-98.
7Jose S, Prema MT, Chacko AJ, Thomasa AC, Souto EB. Colon specific chitosan microspheres for chronotherapy of chronic stable angina. Colloids Surf B Biointerfaces 2011;83:277-83.
8Shigehiro O. Chronotherapeutic strategy: Rhythm monitoring, manipulation and disruption. Adv Drug Deliv Rev 2010;62:859-75.
9Smolensky MH, Lemmer B, Reinberg AE. Chronobiology and chronotherapy of allergic rhinitis and bronchial asthma. Adv Drug Deliv Rev 2007;59:852-82.
10Portaluppi F, Lemmer B. Chronobiology and chronotherapy of ischemic heart disease. Adv Drug Deliv Rev 2007;59:952-65.
11Burioka N, Fukuoka Y, Koyanagi S, Miyata M, Takata M, Chikumi H, et al. Asthma: Chronopharmacotherapy and the molecular clock. Adv Drug Deliv Rev 2010;62:946-55.
12Srinivasan K, Muruganandan S, Lal J, Chandra S, Tandan SK, Prakash VR. Evaluation of anti-inflammatory activity of Pongamia pinnata leaves in rats. J Ethanopharmacol 2001;78:151-7.
13Das KD. Naturally occurring flavonoids: Structure, chemistry, and high-performance liquid chromatography methods for separation and characterization. Methods Enzymol 1994;234:410-20.
14Holiman PC, Hertog MG, Katanc MB. Analysis and health effects of flavonoids. Food Chem 1996;57:43-6.
15Mohammad A. The role of fruits, vegetables and spices in diabetes. Int J Nutr Pharmacol Neurol Dis 2011;1:27-35.
16Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry 2000;55:481-504.
17Hou YC, Chao PD, Ho HJ, Wen CC, Hsiu SL. Profound difference in pharmacokinetics between morin and its isomer quercetin in rats. J Pharm Pharmacol 2003;55:199-203.
18Windholz M. The Merck Index. 11 th ed. United States: Merck and Co., Inc; 1989. p. 986-7.
19Wijeratne SS, Abou-Zaid MM, Shahidi F. Antioxidant polyphenols in almond and its coproducts. J Agric Food Chem 2006;54:312-8.
20Aggarwal BB, Shishodia S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 2006;71:1397-421.
21Lotito SB, Frei B. Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: Cause, consequence, or epiphenomenon?. Free Rad Biol Med 2006;41:1727-46.
22Xie MX, Long M, Liu Y, Qin C, Wang YD. Characterisation of the interaction of human serum albumin and morin. Biochim Biophys Acta 2006;1760:1184-91.
23Francis AR, Shetty TK, Bhattacharya RK. Modulating effect of plant flavonoids on the mutagenicity of N-methyl-N-nitro-N-nitrosoguanidine. Carcinogenesis 1989;10:1953-5.
24Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic Biol Med 1994;16:845-50.
25Fang SH, Hou YC, Chang WC, Hsiu SL, Chao PD, Chiang BL. Morin sulfates/glucuronides exert anti-inflammatory activity on activated macrophages and decreased the incidence of septic shock. Life Sci 2003;74:743-56.
26Cook NC, Samman SJ. Flavonoids: Chemistry, metabolism, cardioprotective effects and dietary sources. Nutr Biochem 1996;7:66-76.
27Middleton JR, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease and cancer. Pharmacol Rev 2000;52:673-751.
28Brown J, O'Prey J, Harrison PR. Enhanced sensitivity of human oral tumours to the flavonol, morin, during cancer progression: Involvement of the Akt and stress kinase pathways. Carcinogenesis 2003;24:171-7.
29Yu ZF, Fong WP, Cheng CH. The dual actions of morin (3, 5, 7, 2', 4'-penta-hydroxyl flavone) as a hypouricemic agent: Uricosuric effect and xanthine oxidase inhibitory activity. J Pharmacol Exp Ther 2006;316:169-75.
30Cao J, Boucher W, Theoharides TC, Kempuraj D, Madhappan B, Christodoulou S. Flavonoids inhibit proinflammatory mediator release, intracellular calcium ion levels, and protein kinase C theta phophorylation in human mast cells. Br J Pharmacol 2005;145:934-44.
31Kuo HM, Chang LS, Lin YL, Lu HF, Yang JS, Lee JH, et al. Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway. Anticancer Res 2007;27:395-406.
32Hodek P, Trefil P, Stiborova M. Flavonoids-potent and versaltile biologically active compounds interacting with cytochromes P450. Chem Biol Interact 2002;139:1-21.
33Lee SF, Lin JK. Inhibitory effects of phytopolyphenols on TPAinduced transformation, PKC activation, and c-Jun expression in mouse fibroblast cells. Nutr Cancer 1997;28:177-83.
34Iwase Y, Takemura Y, Juichi M, Mukainaka T, Ichiishi E, Ito C, et al. Inhibitory effect of flavonoid derivatives on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors. Cancer Lett 2001;173:105-9.
35Kawabata K, Tanaka T, Honjo S, Kakumoto M, Hara A, Makita H, et al. Chemopreventive effect of dietary flavonoid morin on chemicallyinduced rat tongue carcinogenesis. Int J Cancer 1999;83:381-6.
36Cheng CH. In vitro and in vivo inhibitory actions of morin on rat brain phosphatidylinositolphosphate kinase activity. Life Sci 1997;61:2035-47.
37Essa MM, Subramanian P. Pongamia pinnata modulates oxidant- antioxidant imbalance during hyperammonemic rats. Fund Clin Pharm 2006;3:299-303.
38Subash S, Subramanian P, Sivaperumal R. Antihyperammonemic effect of morin: A dose dependent study. J Cell Tissue Res 2007;7:1043-6.
39Wolheim DF. Preanalytical increase of ammonia in blood specimens from healthy subjects. Clin Chem 1984;30:906-8.
40Varley H, Gowenlock AH, Bell M. Practical Clinical Biochemistry. 4 th ed. United States: CBS Publishers; 1998. 1:161-10.
41Reitman S, Frankel AS. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic ransaminases. Am J Clin Pathol 1957;28:56-63.
42King E, Armstrong AR. Determination of serum and bile phosphatase activity. Canadian Med Assoc J 1934;31:376-8.
43Nichans WG, Samuelson B. Formation of MDA from phospholipids arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-30.
44Jiang ZY, Hunt JV, Wolff SP. Detection of lipid hydroperoxides using the 'Fox method'. Annal Biochem 1992;202:384-9.
45Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of SOD. Indian J Biochem Biophys 1984;21:130-2.
46Sinha KA. Colorimetric assay of catalase. Annal Biochem 1972;47:389-94.
47Rotruck JJ, Pope AL, Ganther HE, Swanson AB. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179: 588-90.
48Bradely DW, Hornebeck CL. Clinical evaluation of an improved TFA micro method for plasma and serum vitamin A. Biochem Med 1973;7:78-86.
49Baker H, Frank O, De angelis B, Feingold SE. Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr Rep Int 1980;21:521-6.
50Roe JH, Kuether CA. Detection of ascorbic acid in whole blood and urine through the 2,4-dinitrophyenyl hydrazine of dehydroascorbic acid. J Biol Chem 1943;147:399-407.
51Ellman GL. Tissue sulfhydrl groups. Arch Biochem Biophys 1959;82:70-7.
52Nelson DL, Cox MM. Lehninger Principles of Biochemistry. London: Macmillan; 2000.
53Subash S, Subramanian P. Effect of morin on the levels of circulatory liver markers and redox status in experimental chronic hyperammonaemic rats. Singapore Med J 2008;49:650-5.
54Subash S, Subramanian P. Morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: A biochemical and histopathological study. Mol Cell Biochem 2009;327;153-61.
55Beal MF. Role of excitotoxicity in human neurological disease. Curr Opin Neurobiol 1992;2:657-62.
56Kosenko E, Kaminski Y, Lopata O, Muravyov N, Felipo V. Blocking NMDA receptors prevents the oxidative stress induced by acute ammonia intoxication. Free Radic Biol Med 1999;26:1369-74.
57Choi DW. Ionic dependence of glutamate neurotoxicity. J Neuro Sci 1987;7:369-79.
58Manev H, Favaron M, Guidotti A, Costa E. Delayed increase of Ca influx elicited by glutamate: Role in neuronal death. Mol Pharmacol 1989;36:106-12.
59Fedele E, Jin Y, Varnier G, Raiteri M. In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway. Br J Pharmacol 1996;119:590-4.
60Hermenegildo C, Monfort P, Felipo V. Activation of N-methyl-Daspartate receptors in rat brain in vivo following acute ammonia intoxication: Characterization by in vivo brain microdialysis. Hepatol 2000;31:709-15.
61Hensley K, Tabatabaie T, Stewart CA, Pye Q, Floyd RA. Nitric oxide and derived species as toxic agents in stroke, AIDS dementia, and chronic neurodegenerative disorders. Chem Res Toxicol 1997;10: 527-32.
62Norenberg MD, Rama Rao KV, Jayakumar AR. Ammonia neurotoxicity and the mitochondrial permeability transition. J Bioenerget Biomemb 2004;36:303-7.
63Affany A, Salvayre R, Douste-Blazy L. Comparison of the predictive effect of various flavonoids against lipid peroxidation of erythrocyte membranes (induced by cumen hydroperoxide). Fund Clin Pharm 1987;1:451-7.
64Kok LD, Wong YP, Wu TW. Morin hydrate A potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs. Life Sci 2000;67:91-9.
65Gottlieb M, Leal-Campanario R, Campos-Esparza MR. Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia. Neurobiol Dis 2006;23:374-86.
66Vidhya M, Subramanian P. Enhancement of circulatory antioxidants by alpha-ketoglutarate during sodium valproate treatment in Wistar rats. Polish J Pharmacol 2003;55:31-6.
67Devipriya S, Shyamaladevi CS. Protective effect of quercetin in cisplastin- induced cell injury in the rat kidney. Ind J Pharmacol 1999;31:422-6.
68Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933-56.
69Selvaraju S, Perumal S. Morin improves the expression of urea cycle enzymes in hyperammonemic rats. J Pharm Res 2010;3:2557-60.
70Selvaraju S, Perumal S. Impact of morin (a bioflavonoid) on ammonium chloride-mediated oxidative damage in rat kidney. Int J Nutr Pharmacol Neurol Dis 2011;1:174-8.
71Halberg F, Cornélissen G, Wang Z, Wan C, Ulmer W, Katinas G, et al. Chronomics: Circadian and circaseptan timing of radiotherapy, drugs, calories, perhaps nutriceuticals and beyond. J Exp Ther Oncol 2003;3:223-60.
72Singh R, Singh RK, Singh RK, Tripathi AK, Cornflissen G, Schwartzkopff O, et al. Chronomics of circulating plasma lipid peroxides and antioxidant enzymes and other related molecules in cirrhosis of liver. Biomed Pharmacother 2005;59:229-35.
73Haus E, Pulford DJ, Touitou Y. Chronobiology in laboratory medicine. In: Biologic rhythms in clinical and laboratory medicine. Touitou Y, Haus E, editors. Berlin, Heidelberg, New York: Springer-Verlag; 1992. p. 673-708.