Stress is defined as a nonspecific response of the body to any demand imposed
on it. Stress represents a reaction of the body to a stimulus that tends to
alter its normal physiological equilibrium or homeostasis. Every individual
is likely to face stressful situations in day to day life (Rai
et al., 2003). When stress becomes extreme it is harmful for the
body and hence need to be treated. Stress is involved in pathogenesis of a variety
of diseases and psychiatric disorder such as depression, anxiety, immunosuppressant
and endocrine disorder including diabetes mellitus, male impotence, cognitive
dysfunction, peptic ulcer, hypertension, ulcerative colitis (Singh
et al., 2009) and influence on eating behavior in the humans (Gupta
et al., 2009). Stress is a complex array of internal and external
factors which influences on the appetite and consequently increases the amount
and types of food intake by humans. When an acute stress is experienced, such
as a threat to personal safety, there is an instant physiologic response flight
or fight which results in the suppression of appetite. Exposure to chronic
psychological stressors e.g., job pressure is reason for one of many mental
health disorders that contribute to the global burden of stress associated disease.
In the chronic stressful situations rather than avoiding food, individual seeks
to consume energy dense foods. If stress causes some individuals to consume
food in excess than requirement, this may culminate in weight gain and contribute
to development of obesity. Internal factors have physiologic mechanisms that
regulate appetite by hormone release such as neuropeptide-Y which stimulates
food intake and hormone l Eptin reduce food intake (Torres
and Nowson, 2007).
Chronic stress is one of the etiologies for obesity, the underlying physiological
mechanisms explains potential link between stress and abdominal obesity. The
release of glucocorticoids (e.g., cortisol) due to stress response may disrupt
the food intake regulation in humans by stimulating the neuropeptide Y system
(food intake stimulation) and blunting the effect of the l eptin system (food
intake reduction) which leads to obesity (De Vriendt et
Obesity is a major cause of morbidity and mortality. The prevalence of obesity
(BMI = 30) continues to be a health concern in individuals. Approximately 1.2
billion people in the world are overweight and at least 300 million of them
are obese. According to the World Health Organization (WHO), obesity is one
of the 10 most preventable health risks (Wolf and Colditz,
Obesity is becoming an increasingly great threat to human health. Obesity affects
nine organ systems and a major risk factor for gastroesophageal reflux disease,
nonalcoholic fatty liver disease, cholelithiasis and colon cancer. The primary
environmental determinants of obesity are high calorie intake and decrease in
physical activity. It is a multi factorial disorder, which is often associated
with a considerable burden of ill health and other disorders such as diabetes;
hypertension associated cardiovascular diseases, osteoarthritis and cancer (Liu
et al., 2010).
As there is an increased scientific research in herbals, it is clear that the
medicinal herbs are equally potential in todays synthetic era. According
to various studies its evident that only 20% of the plant flora has been
studied and 60% of synthetic medicines owe their origin to plants. Hence, all
over the world, there is increase in search of natural system of healing (Sarah
et al., 2000).
The available synthetic antistress drugs produce severe adverse effects like
headaches, dry mouth, insomnia respiratory disturbance, constipation and trigger
systemic arterial hypertension associated with cardiac arrhythmias. Herbs can
be used on a long-term basis without a risk of serious side effects (Sarah
et al., 2000).
Man approaches are put forth for treatment of stress, American ginseng is found
to reduce stress, lower high blood sugar and adjust immunity. Various studies
have shown that Ginsenoside, saponin, glycosides and flavonoid constituents
of Panax quinquefolius L. (Nocerino et
al., 2000) and Withania somnifera plant are responsible for adaptogenic
The Apium graveolens extracts were traditionally used for diabetes,
headache, hepatic disorder (Singh and Handa, 1995),
hypercholesterolaemic (Tsi and Tan, 2000), stomach
ache, CNS stimulant, antimicrobial (Momin and Nair, 2001),
antioxidant (Momin and Nair, 2002), anitinflammatory,
inflammation and pain (associated with sprains, bruises, wounds, spasmodic colics
and rheumatic arthritis (Lewis et al., 1985).
This study was planned to screen adaptogenic of Apium graveolens having
the similar chemical constituents as of Withania somnifera, Panax quinquefolius
L., V. vinifera, Bacopamonnieri (Rai et
al., 2003), Zinziber officinalis, Piper nigrum, Emblica officinalis
and Terminalia bellerica (Guevara et al.,
MATERIALS AND METHODS
Extraction procedure: The fresh leaves were thoroughly washed and
dried in shade. The dried leaves were crushed to coarse powder using a hand
mill and sieved (sieve No. 10). Coarse powder was successively extracted in
soxhlet apparatus with 70% ethanol (60-80°C) for 24 h and cold maceration
with distilled water and added few milliliter of chloroform to avoid fungal
growth in aqueous extract. The extracts were concentrated by evaporating them
at room temperature and air dried. The above obtained extracts of ethanolic
and aqueous extracts were dissolved in distilled water (vehicle). All preparations
(doses) were freshly prepared on the day of experiment and administered to the
Preliminary phytochemical analysis: Preliminary phyto-chemical studies
were carried out for all the two different extracts of Apium graveolens
to find out the presence of different phyto-chemical constituents like carbohydrates,
proteins, fats and oil, alkaloids, glycosides, terpenoids, flavonoids, tannins
and polyphenols (Liu et al., 2008; Hu
et al., 2008).
Animal studies: Swiss albino mice of either sex (25-35 g) or male wistar rats (200-250 g) were used. The animals were obtained from Drug Testing Laboratory (DTL) Bangalore, Karnataka. Animals were maintained in suitable nutritional and environmental condition throughout the experiment. The animals were acclimatized for 10 days under standard laboratory condition i.e., room temperature 25±2°C, relative humidity 65±10%, 12 h light/dark cycle. They were fed with standard pellet diet (Venkateshwara enterprises, Bangalore) and water ad libitum under hygienic condition.
Acute toxicity studies (Klein et al., 2007):
Acute oral toxicity studies were carried out for ethanolic and aqueous extracts
of Apium graveolens using acute toxic class method according
to OECD guidelines No. 423. Healthy adult Swiss albino mice (female) weighing
between 25 to 35 g was used for the study. Animals were divided in to four groups
of three animals each, fasted overnight and administered different doses 5,
50, 300, 2000 mg kg-1 b.wt. to each group of animals. The physiological
changes in these animals like body temp, CNS activities, micturition and defecation
etc were observed for 24 h.
Swimming endurance test (Rangari, 2002): Healthy
adult Swiss albino mice of either sex weighing between 25 to 35 g were divided
in to six groups of six animals each. Group I animals served as control were
administered vehicle alone (1 mL/100 g, p.o.). Group II and III animals were
administered aqueous extract of Apium graveolens at dose of 200 and 400
mg kg-1, respectively and Group IV and V animals were administered
ethanolic extracts of Apium graveolens at dose of 200 and 400 mg kg-1.
Group VI animals were administered standard drug (Withania somnifera extract
100 mg kg-1). All the extracts were administered orally once daily
for 7 days and allowed for free access to food and water. On 7th day 1 hr after
extract treatment, all the mice were subjected to swimming endurance test. The
mice were allowed to swim individually in a propylene tank of dimension 37x37x30
cm, filled with water to a height of 25 cm maintained at 26±1°C temperatures.
The end point was death of animals due to drowning when the animals remained
at the bottom of swimming tank. The mean swimming time for each group was calculated.
Anoxia stress tolerance test (Rangari, 2002):
Healthy adult Swiss albino mice of either sex weighing between 25 to 35 g were
divided in to six groups of six animals each. Group I animals served as control
were administered vehicle alone (1 mL/100 g, p.o.). Group II and III animals
were administered aqueous extract of Apium graveolens at dose of 200
and 400 mg kg-1, respectively and Group IV and V animals were administered
ethanolic extracts of Apium graveolens at dose of 200 and 400 mg kg-1.
Group VI animals were administered standard drug (Withania somnifera extract
100 mg kg-1). All the extracts were administered orally once daily
for three weeks and were allowed for free access to food and water. At the end
of 1st week (7th day), 2nd week (14th day) and 3rd week (21st day) week 1 h
after the extract treatment, stress was induced in all mice by placing each
animal individually in the hermetic vessel of 500 mL capacity to record anoxia
tolerance time. The moment when the animal showed the first convulsions, it
is was immediately removed from the vessel and resuscitated if needed. The time
duration of animal entry of the animal into the hermetic vessel and the appearance
of the first convulsion was taken as time of anoxia tolerance. Appearance of
convulsion was very sharp end point, as delay by minute of removal of the animal
from the vessel may lead to death of the animal.
Cold restraint stress (Rangari, 2002): Healthy
adult Wistar rats were divided in to six groups of six animals each. Group I
animals served as control were administered vehicle alone (1 mL/100 g, p.o.).
Group II and III animals were administered aqueous extract of Apium graveolens
at dose of 200 and 400 mg kg-1, respectively and Group IV and
V animals were administered ethanolic extracts of Apium graveolens at
dose of 200 and 400 mg kg-1. Group VI animals were administered standard
drug (Withania somnifera extract 100 mg kg-1). The extracts
were administered orally once daily to respective groups for 10 days. All the
animals were subjected to cold stress by exposing them to 4±1°C daily
for 2 h. This procedure was repeated for 10 days between 11.00 am to 1.00 pm.
On 10th day blood was collected from the hepatic portal vein under light ether
anesthesia to estimate biochemical parameters like serum glucose, cholesterol,
triglyceride and BUN (Blood Urea Nitrogen). Animals were sacrificed by cervical
dislocation and the weight of organs such as liver, spleen, testes and adrenal
gland were recorded per 100 g body weight of animal after washing them with
alcohol (Hu et al., 2008).
Statistical analysis: Data obtained were expressed as Mean±Standard error of mean (SEM). The significance was determined by applying one-way ANNOVA using prism graph pad software. The statistical differences in the sample means were considered significant at p<0.05.
The preliminary phytochemical analysis of the ethanolic and aqueous extracts
of Apium graveolens revealed the presence of the phyto-chemical constituents
like alkaloids, carbohydrates (Reducing sugars Non-reducing Sugar), Glycosides
(Coumarin Saponin Phenolic and Flavonoid), flavanoids, proteins and aminoacids
in both additionally ethanolic extract contains tannins, fats, terpenoids, tannins
Acute oral toxicity studies of the ethanolic and aqueous extracts of Apium graveolens did not exhibit any sign of toxicity up to 2000 mg kg-1 b.wt. Since, there was no mortality of the animals found at highest dose. In the present study dose was selected randomly i.e., 200 and 400 mg kg-1.
It was found that ethanolic and aqueous extracts of Apium graveolens induced a striking increase in swimming endurance time in mice was found to be dose dependant. The extract shows significant increase in the percentage increase in swimming endurance time over vehicle treated animals by 26.82% in 200 mg kg-1 and 38.05% in 400 mg kg-1 of aqueous extracts of Apium graveolens and 48.65% in 200 mg kg-1 and 52.70% in 400 mg kg-1 of ethanolic extracts of Apium graveolens in the treated groups, respectively. Result are expressed as Mean±SEM (Table 1).
The effect of aqueous and ethanolic extracts of Apium graveolens on
anoxia stress tolerance test in mice was found to be dose dependant increase
(Table 2). Treatment with aqueous extract of 200 and 400 mg
kg-1 has produced stress tolerance time 33.9±1.38 and 36.65±1.31
in 1st h, 39.76±1.16 and 43.04±1.36 in 2nd h and 46.04±1.21
and 53.59±1.58 in 3rd h. The ethanolic extracts of 200 and 400 mg kg-1
has produced stress tolerance time 34.03±0.27and 37.38±0.3 in
1st h, 41.22±0.7 and 46.12±0.87 in 2nd h and 52.14±0.49
and 61.86±0.62 in 3rd h. Treatment with aqueous and ethanolic extracts
as shown significant increase in anoxia stress tolerance time. The result express
as mean (Table 2).
The chronic stress due to the cold resulted in significant rise in serum glucose, cholesterol, triglycerides and BUN. The aqueous extracts of dose 200 and 400 mg kg-1 as shown reduction in the blood glucose 109.04±3.78 and 102.25±2.04, cholesterol 76.37±1.78 and 74.25±2.04, triglyceride74.61±2.31 and 81.17±4.5 and BUN 25.06±0.67 and 24.58±0.99. Ethanolic extract of dose 200 and 400 mg kg-1 as shown reduction in blood glucose 91.95±1.27 and 84.66±1.57, cholesterol 68.75±2.51 and 55.22±1.61, triglyceride 79±2.01 and 77.73±1.40 and BUN 24.31±0.86 and 24.58±1.50. Concomitant treatment with ethanolic extracts has shown significant and greater extent than aqueous extracts alone in reduction of all the parameter (Table 3). The ethanolic and aqueous extracts were shown significant decrease in the cold stress induce elevated level of serum glucose, cholesterol and triglyceride with respect to Standard (Withaniasomnifera) activity expect BUN.
The chronic stress due to the cold resulted in significant rise of liver, adrenal
gland weight and decrease in spleen and testes weight (Table 4).
The aqueous extracts of dose 200 and 400 mg kg-1 as shown decrease
in the liver weight 7.48±0.18 and 6.30±0.16 and decrease in adrenal
gland weight 0.02±0.00 and 0.027±0.00 and increase in spleen weight
1.16±1.16 and 1.50±0.22 and testes weight 1.60±0.00 1.76±0.00.
Ethanolic extract of dose 200 and 400 mg kg-1 as shown decrease in
the liver weight 6.91±0.31 and 6.30±0.22 and decrease in adrenal
gland weight 0.024±0.00 and 0.023±0.0 and increase in spleen weight
1.00±12.03 and 1.36±0.21 and testes weight 1.60±0.00 and
1.92±0.23. Concomitant treatment with ethanolic extracts has shown Significant
and greater extent than aqueous extracts alone in reduction of all biological
parameters. The ethanolic and aqueous extracts were shown significant decrease
in the cold stress induced increased liver, adrenal gland weight and increase
in weight of spleen and testes respect to Standard activity.
|Table 1: Effect of extract of Apium graveleons on swimming
endurance test in mice. n = 6
|**Significance at p<0.05, ***p<0.001 as compared to
|Table 2: Effect of ethanolic and aqueous extracts of Apium
graveolens on anoxia stress tolerance test in mice n = 6
|Significance at **p<0.01, *p<0.05, ***p<0.001 as
compared to control gp
| Table 3: Effect of ethanolic and aqueous extracts of Apium
graveolens on biochemical parameters of rats in cold restraint stress
n = 6
|Significance at *p<0.05, **p<0.01, ***p<0.001 as
compared to positive control gp (stress rats)
| Table 4: Effect of ethanolic and aqueous extracts of Apium
graveolen on organ weights of rats in cold restraint stress n = 6
|Significance at *p<0.05, **p<0.01***p<0.001, as compared
to positive control group
The swimming endurance test was conducted in mice has shown significant
increase in the swimming endurance time with both doses of ethanolic and aqueous
extracts. The ethanolic extract adaptogenic activity was potential than aqueous
extracts. The activity produced may be possibly due to normalization of plasma
levels of catecholamine and MAO, which are decreased during stress situation
or by decreasing muscle glycogen and preventing accumulation of fat (Friedewald
et al., 1972). The other possible mechanism for this adaptogenic
activity may also be mediated by slowing glycogen depletion, decreases in concentrations
of muscle lactic acid and ammonia. The anti-oxidant property of the Apium
graveolens extracts has already reported and which may also contributes
for the adaptogenic activity.
The anoxia tolerance test was conducted in mice has produced increase in the
anoxia tolerance time with both doses of ethanolic and aqueous extracts. The
anoxia tolerance test of adaptogenic property of the extracts may due to increasing
succinate dehydrogenase (SDH) in the brain. This enzyme is responsible for utilization
and conservation of energy in the cellular system of the organism which helps
adaptive processes during stress (Bhatwadikar et al.,
1999). The effect is probably related to an increase in cerebral resistance
to anoxia and reducing the cerebral consumption of oxygen in anoxic stress.
The present study has given emphasis on estimation of stress induced biochemical
and biological parameters by in vitro method.
Stressful conditions lead to formation of excessive free radicals which are
the major internal treat to cellular homeostasis of aerobic organisms. Free
radicals are formed in human body both in physiological and pathological conditions
in cytosol, mitochondria, lysosomes, peroxisomes and plasma membranes (Patil
et al., 2006). These free radicals are extremely reactive and unstable
chemical species which reacts with proteins, lipids, carbohydrates and nucleic
acids in the body. Exposure of lipids in cell membrane to free radicals stimulates
the process of lipid peroxidation. The free radical activity and the extent
of tissue damage are related quantitatively to the amount of lipid peroxide
level in the blood. There are reports suggesting that there is a stress induced
accelerated lipid peroxidation (Han et al., 2005)
(increased MDA) and alterations in lipid profile affecting serum total cholesterol,
LDL, VLDL and triglycerides. The aqueous and ethanolic extracts were shown significant
reduction in stress induced elevated levels of serum glucose, cholesterol and
triglyceride levels (Kaur and Kulkarni, 2001) except
BUN in a dose of 200 and 400 mg kg-1 with respect to standard (withania
somnifera). Concomitant administration of aqueous extracts and ethanolic
extracts with a dose of 200 and 400 mg kg-1 were shown significant
decrease in liver and adrenal gland weight and increase in weight of spleen
and testes with respect to standard (Withania somnifera).
Both aqueous and ethanolic extracts has produced significant change in the reduction of stress induced biochemical and biological changes in rats. The reduction in the stress induced all the above parameters which may likely to prevent formation of excessive free radicals resulting in preventing internal treat to cellular haemostatis, thus depressing the processes of lipid peroxidation and normalizing the elevated serum glucose, cholesterol and triglycerides.
The ethanolic extract of the plant has produced significant effect in swimming
endurance time, anoxia tolerance time and biochemical and biological parameters
compared to aqueous extracts. Hence, extracts of Apium graveolens considered
to be having potent antistress effect. These extracts might help in preventing
stress induced complications, these results obtained in the study are encouraging
to pursuing further studies on the isolated active constitute present in the
extracts responsible for the activity.
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