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O r i g i n al A r t icle

Chronotherapeutic effect of morin in experimental

chronic hyperammonemic rats
Selvaraju Subash, Perumal Subramanian

Department of Biochemistry ABSTRACT

and Biotechnology, Faculty of
Science, Annamalai University,
Annamalainagar, Tamil Nadu,
India
Address for correspondence:
Dr. Perumal Subramanian,
Department of Biochemistry
and Biotechnology, Faculty of
Science, Annamalai University,
Annamalainagar – 608 002,
Tamil Nadu, India.
E-mail: psub@rediffmail.com
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’,4’-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’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.

Key words: Ammonia, antioxidants, chronotherapy, liver markers, urea

INTRODUCTION important nitrogen substrate in several reactions

and plays an important role in nitrogen homeostasis
Hyperammonemia is defined as elevated ammonia of cells. Moreover, ammonia is a product as well as
concentration in blood, resulting from inadequate precursor of various important nitrogen-containing
ammonia detoxification due to impairment in metabolites such as amino acids, the smallest
liver function. In living organisms, ammonia is an subunits of proteins. [1] Ammonia is neurotoxic
when it accumulates in excess. Hyperammonemia is
Access this article online mainly responsible for the neurological alterations—
Quick Response Code: including impaired intellectual function—found in
Website:
the hepatic encephalopathy seen in patients with
www.ijnpnd.com
liver disease. Antiepileptic drugs such as valproate
DOI:
and salicylate also cause hyperammonemia and urea
10.4103/2231-0738.99483
cycle disorders (UCDs). Ammonia toxicity results in
free radical generation that leads to oxidative stress–

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Selvaraju and Perumal: Chronotherapeutic effect of morin in hyperammonemia
mediated tissue damage,[2–4] 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
International Journal of Nutrition, Pharmacology, Neurological Diseases | September-December 2012 | Vol 2| Issue 3
by two aromatic rings, ring A and ring B, joined by a
three-carbon-linked c-pyrone ring (ring C), forming a
C6–C3–C6 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,[23–25] 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 chro-
notherapeutic effect of morin on circulatory (i) am-
monia, urea, alanine transaminase (ALT), aspartate
transaminase (AST), and alkaline phosphatase (ALP);
(ii) thiobarbituric acid reactive substances (TBARS) and
hydroperoxides (HP); and (iii) enzymatic and nonen-
zymatic 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
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Selvaraju and Perumal: Chronotherapeutic effect of morin in hyperammonemia

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±2ºC 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.
RESULTS
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.
The levels of circulatory antioxidants in control

Table 1: Chronotherapeutic effect of morin on blood ammonia, plasma urea, TBARS, HP, and serum AST,
ALP, and ALT of normal and experimental rats
Groups Blood ammonia plasma urea TBARS HP Liver marker enzymes
(µmol/L) (mg/dL) (nmol/mL) (×10-5 mmol/dL) AST ALP ALT
(IU/L) (IU/L) (IU/L)
Normal 83.81 ± 6.38a 9.99 ± 0.76a 2.72 ± 0.21a 8.08 ± 0.62a 69.35 ± 5.28a 70.55 ± 5.37a 23.44 ± 1.79a
Morin (30 mg/kg) 80.16 ± 6.14a 9.08 ± 0.70a 2.70 ± 0.21a 8.10 ± 0.62a 68.69 ± 5.26 a
70.47 ± 5.39 a
22.79 ± 1.74a
AC (100 mg/kg) 376.48 ± 28.67b 24.97 ± 1.90b 4.72 ± 0.36b 14.12 ± 1.08b 121.15 ± 9.23 139.67 ± 10.64
b b
65.57 ± 4.99b
AC + morin (06:00 h) 193.80 ± 14.80c 15.14 ± 1.16c 3.81 ± 0.29c 12.45 ± 0.95c 111.25 ± 8.50c 112.25 ± 8.57c 38.45 ± 2.94c
AC + morin (12:00 h) 152.77 ± 11.67d 13.77 ± 1.05d 3.45 ± 0.26d 10.23 ± 0.78d 93.26 ± 7.12d 90.48 ± 6.91d 31.22 ± 2.38d
AC + morin (18:00 h) 200.12 ± 15.24c 15.41 ± 1.17c 3.80 ± 0.29c 12.65 ± 0.96c 110.16 ± 8.39c 114.34 ± 8.71c 37.78 ± 2.88c
AC + morin (24:00 h) 125.55 ± 9.59e 11.55 ± 0.88e 3.10 ± 0.24e 9.23 ± 0.71e 80.55 ± 6.15e 80.03 ± 6.11 27.79 ± 2.12e
ANOVA followed by Duncan’s multiple range test, Values not sharing a common superscript (a, b, c) differ significantly at P≤0.05

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Selvaraju and Perumal: Chronotherapeutic effect of morin in hyperammonemia

Table 2: Chronotherapeutic effect of morin on changes in the enzymatic and non-enzymatic antioxidants
of normal and experimental rats
Groups SODA CATB GPxC Vitamin A in Vitamin E in Vitamin C in GSH in
plasma plasma plasma plasma
(mg/dl) (mg/dl) (mg/dl) (mg/dl)
Normal 2.81 ± 0.21a 2.10 ± 0.16a 24.23 ± 1.85a 2.18 ± 0.17a 1.58 ± 0.12a 1.59 ± 0.12a 26.89 ± 2.05a
Morin (30 mg/kg) 2.83 ± 0.22a 2.11 ± 0.16a 24.57 ± 1.88a 2.25 ± 0.17a 1.59 ± 0.12a 1.60 ± 0.12a 27.12 ± 2.08a
AC (100 mg/kg) 1.70 ± 0.13b 1.32 ± 0.10b 12.76 ± 0.97b 1.41 ± 0.11b 0.91 ± 0.07b 0.93 ± 0.07b 14.23 ± 1.08b
AC + Morin (06:00 h) 2.10 ± 0.16c 1.50 ± 0.11c 15.79 ± 1.21c 1.65 ± 0.13c 1.05 ± 0.08c 1.18 ± 0.09c 18.34 ± 1.40c
AC + Morin (12:00 h) 2.33 ± 0.18d 1.75 ± 0.13d 19.02 ± 1.45d 1.83 ± 0.12d 1.22 ± 0.09d 1.31 ± 0.10d 21.22 ± 1.62d
AC + Morin (18:00 h) 2.11 ± 0.16c 1.51 ± 0.11c 16.11 ± 1.23c 1.66 ± 0.13c 1.04 ± 0.08c 1.19 ± 0.09c 18.55 ± 1.41c
AC + Morin (24:00 h) 2.57 ± 0.20e 1.92 ± 0.15e 21.35 ± 1.63e 2.01 ± 0.15e 1.38 ± 0.11e 1.45 ± 0.11e 24.24 ± 1.85e
ANOVA followed by Duncan’s multiple range test. Values not sharing a common superscript (a, b, c) differ significantly at P ≤ 0.05, AAmount of enzyme required
to inhibit 50% of NBT reduction/mg Hb, BMicromoles of H2O2 consumed/min/mg Hb, CMicromoles of GSH utilised/gHb

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.
DISCUSSION
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 Ca2+ concentration in the postsynaptic
neuron.[57,58] Ca2+ 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
disability.[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

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Selvaraju and Perumal: Chronotherapeutic effect of morin in hyperammonemia
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]
CONCLUSION
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.
ACKNOWLEGMENT
Financial assistance from the University Grants Commission,
New Delhi, is gratefully acknowledged.
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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;2:266-71.
Source of Support: University Grants Commission. Conflict of
Interest: None declared.
Received: 06-09-2011, Accepted: 23-11-2011
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