To Study the Effect of Allium sativum on various Biochemical Parameters on Stress Induced in Albino Rats

 

Roshan S, Tazneem B, Abdullah Khan and Sadath Ali*

Luqman College of Pharmacy, Gulbarga, Karnataka.

ABSTRACT:

The present study was carried out to evaluate the effect of ethanolic extract of Allium sativum on swimming endurance test and cold stress in albino rats. The effect was assessed by swimming survival time, estimation of various biochemical parameters like glucose, cholesterol, triglycerides, cortisol and blood urea nitrogen (BUN). Blood cell count (WBC) and also the differential count was measured. In cold stress ulcer index , PH­of gastric juice and weight of organ such as liver, spleen, testes, adrenal gland at a dose of and 200 mg/kg body weight per oral was determined. It was found that extract significantly (p<0.001) increases swimming time. Showed significant (p<0.001) decrease in blood glucose, cholesterol, triglyceride, cortisol and BUN. A significant (p<0.01) decrease in WBC count, polymorphs, monocytes, lymphocytes and eosinophils in swimming endurance test.  In cold stress significant (p<0.01) decrease in weight of organs, significant (p<0.001) decrease in ulcer index and increase PH ­of gastric juice was observed compared to control group. Thus the obtained results revealed that the Allium sativum has got a significant anti-stress activity.

 

KEYWORDS: Swimming, Cold stress, Allium sativum, WBC, BUN

 

INTRODUCTION:

Hans Selye, has defined stress as the sum of all the non-specific changes caused by function or damage. It is fundamentally a physiological response, the primary object of which is to maintain life and re-establish the normal state. The general manifestations of stress are called the ‘General adaptation syndrome’ (G.A.S.), which comprises of 3 stages. Alarm reaction, resistance and exhaustion, respectively1

 

Fight or flight, alarm phase: This response is an alarm reaction triggered by messages in the brain. The pituitary gland releases adrenocorticotropic hormone (ACTH). ACTH causes the adrenal glands to secrete adrenaline, cortisol, and other stress hormones2. The heart beats faster to provide blood to the muscles and brain. The breath rate increases to supply extra oxygen to the muscles, heart, and brain. Digestion and other functions not essential for maintaining the alarm reaction are halted. The liver rids itself of stored glycogen and releases glucose into the bloodstream. The body is now ready for any real or imagined danger.

 

Adaptation phase: If the stress factor continues (for example, in sport it might be heavy athletic training), our body learns to tolerate the stressful stimulus “adapt” and increase its resistance to the stress factor. The adaptation phase is usually a safe period and allows the body to endure ongoing stress3. The longer we can stay in the adaptation phase, the better.

 

Exhaustion phase: In this phase, the body fails to fight stress anymore and simply gives up. This stage is a result of chronic over secretion of cortisol. This leads to adrenal exhaustion. Adrenal exhaustion accelerates the downward spiral to chronic poor health. Chronic headaches, nausea, allergies, nagging injuries, fatigue, dizziness, hypotension, low body temperature, depression, low sex drive, chronic infections, and cold hands and feet are just some of the symptoms that occur with adrenal exhaustion4.

 

The bulb Allium sativum belongs to family Alliaceae, perineal bulbous scapigerous herb. It is cultivated throughout India.  It has great medicinal value; bulb used has anti diabetic, anti-cancer, anti hypertensive, and lipidemic activity. The juice is effective in rheumatism, whooping cough, tuberculosis and has anti bacterial and rejuvenative actions. The fresh bulb is used as rejuvenative. The plant contains proteins, amino acids, flavonoids, hydrocarbons, allin, sulphur amino acid, anthocyanins, kaempferal and carbohydrates5. The plant reported to have anti diabetic6, anti-oxidant7, hepataprotective8, anti stress9 and anti lipidemic10 effects. In the present study ethanolic extract of Allium sativum was evaluated for its effects on adaptogenic activity.

 

MATERIALS AND METHOD:

The bulb Allium sativum was collected from local area of Gulbarga, and Authentificaton was done by Dr. Seetharam. G.   Department of botany, Gulbarga University, Gulbarga. A voucher specimen number (LCP 216). The same has been deposited in research laboratory. Ethanolic extract Panax ginseng a gift sample obtained from Madhur Pharma, Bangalore (PAN-C00531). Fresh bulb crushed and pulverized in laboratory, extracted with 95% ethanol in percolator. The extract was concentrated by using rotary flash evaporator. The residue was dried in desicator over sodium sulphate. The dried ethanolic extract was suspended in distill water using 1% Tween 80 used for pharmacological screening.

 

Experimental animals:

Adult Swiss albino mice (20- 25g) and Wistar rats (150 -200g) of either sex were used for the study. The mice and rats were fed with standard pellet (Parnava Agro industries Ltd. Sangali, India) and water ad libitum. The animals were maintained under standard 12-hr light / dark cycle throughout the study. The study protocol was approved by IAEC. (No.CPCSEA/IAEC/PC-01/346)

 

Acute toxicity study:11

The study was performed according to the acute toxic classic method (as per CPCSEA/OECD guidelines). Swiss albino mice were used for acute toxicity study. The animals were kept Fasting for overnight providing only water, after which the test drug extract dissolved in Water was administered orally at the dose of 800 mg/kg and observed for 14 days. Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 h (with special attention during the first 4 h) and daily thereafter for a period of 14 days. Once daily cage side observations included changes in skin and fur, eyes and mucous membrane (nasal) and also respiratory rate, circulatory(heart rate and blood pressure), autonomic (salivation, lacrimation, perspiration, piloerection urinary incontinence, and defecation) and central nervous system (ptosis, drowsiness, gait, tremors and convulsion). The toxicity study carried out as per the guidelines of AOT- 421 using albino mice. The extracts were found to be safe till 400mg/kg. Hence we selected 200mg/kg dose for pharmacological screening.

 

Swim endurance test:12

Albino rats (120 ± 150 g) of either sex were randomly divided into 4 groups of 6 animals each. Group I control, Group II swimming test  control, Group III swimming test  and subjected to the oral administration of extract Allium sativum at the dose 200 mg/kg and Group IV subjected to swimming test and the oral administration of Panax ginseng extract at the dose 100 mg/kg p.o respectively for 21 days. Swimming test was carried out on 7th, 14th and 21stday . One hour after the drug administration mice were allowed to swimming in (24×24×18 cm) rectangular container filled with water maintained at 25 ± 2°C till they got exhausted and the moment they drowned head was considered as the endpoint the time was noted13. On 21stday all the animals were sacrificed and blood was collected for estimation of biochemical parameters like, glucose, cholesterol, triglycerides, BUN and plasma cortisol14, blood cell count15 the data obtained were subjected to statistical analysis.

 

Cold stress:

Albino rats (150-180gm) of either sex were divided in to 4 groups of 6 animals each. Group -I served as control, Group-II served as cold stress control , Group-III served as cold stress induce and Allium sativum extract 400mg/kg  p.o, and  group - IV cold stress induce and Panax ginseng  100mg/kg p.o.  Cold Stress was induced in 2nd, 3rd and 4th  groups in albino rats , by exposing animals to 4 ±1°C daily for 2 hrs for10 days15. On 11th day all the animals were sacrificed and remove the organs such as liver, spleen, testes and adrenal gland note weight of organ16.The stomach was removed and split open along the greater curvature. The numbers of discrete ulcers were noted by the help of magnifying glass.17

 

The animals were dissected and the stomach carefully keeping the esophagus closed opened along the greater curvature the gastric contents were collected in a tube and centrifuged at 3000 rpm for 5 min, the volume of supernatant was expressed as ml/100g body weight. The mucosa was flushed with saline finally the ulcers were observed macroscopically. The observation was made for any ulceration or inflammation in the stomach. The stomachs were opened along the greater curvature and the mucosa was exposed for evaluation. Ulcer index, percentage protection and the P H of gastric juice was determined statistically18.

Statistical analysis:

Analysis of data was performed using statistical package for social version 11.0 (SPSS) computer software.  Descriptive statistics were adopted to display data in mean of ± SEM ANOVA was used to compare the mean value obtained between the different groups.  Differences were considered significant whenever the P value are reported as mean ± SEM. ***p<0.001, **p<0.01 and *p<0.05.

 

RESULTS:

In Swimming endurance test ethanolic extract of Allium sativum at a dose of 200mg/kg b.w has shown significantly (p<0. 001) increased in the swimming time as compared to control (Table 1)

 

Table 1 Effect of Allium sativum on swimming endurance test.

Group

Mean duration of Swimming survival time (in min.) Mean± SEM

1st week

2nd week

3rd week

Control

32.3 ±1.91

49.8 ±4.11

64.4 ±3.85

Ethanolic extract of

Allium sativum

28.6 ±  1.78**

33.26±3.27**

47.3± 3.28***

Ethanolic extract of panax ginsang

31.4 ± 2.18**

45.8 ± 3.98**

61.6 ± 3.11***

 

In cold stress induced, the ethanolic extract of Allium sativum has significantly (p<0.001) reduced the elevated levels of biochemical parameters glucose, cholesterol, triglyceride, BUN and cortisol levels when compared with stress control group (Table 2). The extract has also reduced the blood cell count WBC’s significantly (p<0.01), except lymphocytes and Eosinophils (p<0.05) compared to stress control group (Table). Determination of weight of organs showed that the extract has significantly (p<0.01) reduced the weight of liver, spleen and testes, however it showed no effect on weight of adrenal gland (Table 3).

 

Cold stress increases the incidence and severity of gastric ulcers. In the present study showed significantly reduced Ulcer incidence (%), increase in PHof Gastric juice (Table 4).

 

DISCUSSION:

Animals when subjected to a period of stress produce characteristic changes in several hormones and parameters associated with central nervous system and hypothalamic-pituitary-adrenal axis (HPA). HPA changes include an increase in cortisol, a reduced sensitivity of the HPA to feedback down-regulation, and a disruption in the circadian rhythm of cortisol secretion. Central nervous system changes include the stress-induced depletion of catecholamine neuro transmitters such as nor epinephrine and dopamine. An acute increase in beta-endorphin levels is also observed under stressful conditions19.

 

Rodents when forced to swim in a restricted space become immobile after an initial period of vigorous activity indicating the stress. Pretreatment with adaptogen increase swimming endurance in rats20. Increase in total swimming time of Allium sativum treated rats showed significant improvement in the swimming time. Stress increases total leukocyte count, eosinophils and basophils. Plant adaptogen are smooth prostressors which reduce the reactivity of host defense system. The mode of action of adaptogens is basically associated with stress system. Adaptogen increase the capacity of stress to respond to the external signals of activating and deactivating mediators of stress response subsequently21,22. The stress induced increase in total WBC count, which is decreased by ethanolic extract of Allium sativum indicating its antistress and adaptogenic activity are similar to the changes produced by reference drug ethanolic extract of Panax ginseng.

 

During stress ACTH is released which acts on adrenal cortex where by cortisol and corticosterone will be secreted. Increased plasma cortisol influences the mobilization of stored fat and carbohydrate reserves, which in turn increases blood glucose level. The increased cortisol levels and increased blood glucose level are reversed by anti-stress agents20-22.

 

A Blood glucose level increase in response to stress is due to release of Glucocorticoids as a result of HPA axis stimulation to compensate initial demand of energy. The acute demand of glucose is fulfilled by the increase in glucogenolysis from liver during stress23, which is found to be significantly reduced Allium sativum and it also significantly reduced stress induced plasma cortisol level exhibiting anti-stress activity which is comparable to standard Panax ginseng.

 

The mechanism by which stress raises serum cholesterol, triglycerides and BUN levels in stress induced animals is due to the enhanced activity of hypothalamo-hypophyseal axis resulting in increased liberation of catecholamines and corticosteroids24. Allium sativum as well as the standard Panax ginseng significantly reduced the elevated serum cholesterol, triglyceride and BUN levels which might be due to the inhibition of stimulation of sympathetic nervous system.

 

The increase in adrenals in stressed animals is due to the stress induced adrenomedullary response leading to increased production of corticotropic hormone that leads to increase in weight of adrenals25. Allium sativum and Panax ginseng has significantly reduced the liver and adrenal gland weight this could be due to the reversal of stress induced adrenomedullary response and hence decrease production of corticotropic hormone. Pretreatment with Abutilon indicum significantly increased the spleen weight. This might be due to the inhibition of recruitment of lymphocytes to blood from spleen.

 

Stress causes alteration in hematological parameters like increase in WBC and DLC counts, neutrophils24. Cold stress typically increases total leukocyte count, eosinophils and basophils25.


Table 2Effect of Allium sativum on biochemical parameter on swimming test stress induced in albino rats.

Parameter

Control

Cold stress

Allium sativum Extract

Panax ginseng Extract

Glucose mg/dL

80.24 ± 0.812

112.2±2.91

96.45±5.21***

86.25±3.11***

Cholesterol mg/dL

40.21±1.882

58.4±1.727

43.29±2.93***

44.54±2.65**

Triglyceride mg/dL

71.24±0.712

105.24±2.64

83.14±1.95***

84.58±1.65**

BUN mg/mL

30.14±0.512

53.41±2.41

39.18±1.22***

34.58±1.87***

Plasma cortisol (μg/100 ml)

13.04 ± 0.24

21.41±0.56*

18.63±0.59*

15.63±0.09**

WBC Cells mm-1

4824 ± 41.02

6872 ± 143.7

5013 ± 14.6***

5217 ±48.21***

Lymphocytes Cells Cumm-1

48 ± 1.72

64 ± 1.09

52 ± 0.52**

59 ± 1.03**

Monocytes Cells Cumm-1

1.00 ± 0.0

3.25 ± 0.19

1.25 ± 0.50**

1.5 ± 0.76**

Neutrophills Cells Cumm-1

24 ± 0.40

35 ± 0.84

25 ± 0.40*

31 ± 0.22**

Eosinophils Cells Cumm-1

1.25 ± 0.4

4.50 ± 0.25

1.0 ± 0.25*

1.0 ± 0.25**

 

 

Table 4 Effect of Allium sativum on weight of organ on cold stress induced in albino rats.

Parameter

Control

Cold stress

Allium sativum extract.

Panax ginseng extract.

Spleen mg/100g

3.57 ±0.11

2.591±0.12

3.08±0.29**

3.38 ± 0.26**

Testes mg/100g

1.681±0.06

1.121±0.02

1.341±0.29**

1.431 ± 0.21**

Liver g/100g

5.142±62.5

6.718±38.2

5.921±13.8***

5.101 ± 10.8**

Adrenal gland  g/100g

0.250±0.01

0.489±0.44

0.358±0.01*

0.318 ± 0.01**

 

 

Table 3 Effect of Allium sativum on gastric ulceration in cold stress induced albino rats.

Parameter

Ulcer index

% Protection

P H of Gastric juice

Control

-

-

6.03± 0.20

Cold stress

12.5 ± 0.7

18.8±3.29

3.0± 0.20

Abutilon indicum extract

5.7 ± .5*

8.8 ± 1.8*

4.9± 0.17*

Panax ginseng extract

4.6 ± 1.44*

3.9±1.01*

5.4± 0.80*

 

 

 

Plant adoptogens are smooth prostressors which reduce the reactivity of host defense system and decrease the damaging effects of various stressors due to increased basal levels of mediators involved in the stress response26. The stress induced increased total WBC count is decreased by ethanolic extract of Allium sativum, thus the plant posses antistress, adoptogenic activity. Allium sativum and Panax ginseng significantly reduced the WBC, lymphocytes, eosinophils and monocyte counts in cold stress.

 

Stress-induced ulcers are caused by number of factors both physical and psychological. Increase in gastric motility, vagal overactivity, mast cell degranulation, decreased mucosal blood flow, and decreased prostaglandin synthesis are reported to be involved in the genesis of stress-induced ulcers27. The result showed the ability and the efficacy of the ethanolic extract of Allium sativum in preventing gastric ulceration in cold stress induced rats suggesting its anti-ulcer activity.

 

A variety of biological activities including Anti-stress activity were reported with flavonoids, tannins and phenolic glycosides. Allium sativum contains biologically active chemicals that include flavonoids, saponins, alkaloids, proteins, fixed oils and proteins. The anti stress activity may be due to the presence of these constituents where as standard drug Panax ginseng an established adaptogenic drug too contains glycosides, steroids and flavoniods.

 

 

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Received on 13.06.2010

Accepted on 17.07.2010

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Research J. Pharmacology and Pharmacodynamics. 2(5): Sept.-Oct. 2010, 335-339