Evaluation of effect of Murraya koenigii on restraint stress induced perturbations

Saraf M.N.¹, Sanaye M.M.²*and Mengi S.A.²

¹Bombay college of pharmacy santacruz (E) Mumbai,Maharashtra,India

²C.U. Shah College of Pharmacy, SNDT University Santacruz (W), Mumbai 400049, Maharashtra, India

ABSTRACT:

Aqueous (MKAQ), Hydroalcoholic (MKHA) and Methanolic extract (MKM) of leaves of Murraya koenigii (MK) at dose levels 50mg/kg 100mg/kg and 200mg/kg p.o. were investigated to gauge antistress activity in fresh wistar rats. Anti anxiety drug Diazepam (1mg/kg p.o.) and natural antistress Ashwagandha (AS) (100mg/kg p.o.) were used as standards. The animals were subjected to restraint stress ( 2 hrs/day) for 14 days to evaluate the anti-stress potential in chronic stress condition. Further they were tested to observe their effects on retrograde amnesia caused by chronic stress in elevated plus maze and Step down inhibitory avoidance tests. Nootropic drug Piracetam (200mg/kg p.o.) was used as a standard for cognition studies. Stimulation of hypothalamus pituitary adrenal axis in stressful condition alters corticosterone , glucose ,triglyceride and cholesterol, levels. There is also alteration in the norepinephrine 5HT and dopamine levels in the brain. Treatment with the extracts significantly ameliorated the stress-induced variations in these biochemical and bioamine levels. Treatment with MKM, MKAQ and MKHA extracts also prevented stress induced adrenomegaly . In cognition studies stress induced increase in latency period and decrease in step down latency in elevated plus maze models and Step down inhibitory avoidance test respectively was prevented. The results indicate that among three test extracts MKM was the most effective extract as compared to MKHA and MKAQ extracts of the plant thus possessing significant antistress potential against a variety of biochemical and physiological perturbations during stress.

KEYWORDS: Chronic restraint stress ,Murraya koenigii, corticosterone , bioamines, antistress

1. INTRODUCTION:

Stress is basically a reaction of mind and body against change in the homeostasis. The productive stress is called Eustress while harmful stress is called Distress. If the stress is extreme, the homeostatic mechanisms of the organism become deficit and the survival of the organism is threatened. Under these conditions, stress triggers a wide range of body changes called General Adaptation Syndrome (GAS). The stimuli, which produce GAS are called the stressors which range from physical to psychological factors including cold, heat, infection, toxins, major personal disappointment¹ etc. In the stress-filled environment we live in, successful adaptation to stress is a prerequisite for survival. In the indigenous system of medicine, there are many herbal drugs and formulations recommended to enable one to withstand stress without altering the physiological functions of the body. Psychological stressors are stimuli that affect emotion and result in fear, anxiety, or frustration, and are among the most potent activators of the HPA axis.^2,3

Stress alters the equilibrium of various hormones namely, the sympathetic nervous system and the hypothalamo–pituitary–adrenal (HPA) axis. The HPA axis along with the sympathoadrenal system governs metabolic responses to the slings and arrows of everyday life, as well as to the beleaguering demands that prevail under conditions of chronic stress. chronic intermittent stress is associated with changes in the adrenal medulla, including increased activity of enzymes involved in catecholamine biosynthesis, increased rate of catecholamine synthesis and elevated tissue concentrations of catecholamines and their utilisation⁴. There is also extensive evidence demonstrating modulatory effects of various stressors on human and animal cognition^5-7 Prior exposure of laboratory rodents to an inescapable stressor often impairs their performance in different learned tasks^8-11 Restraint stress have shown to induce impairment of learning and memory¹².

Thus one can say that stress has been reported to be involved in the etiopathogenesis of a variety of diseases such as psychiatric disorders involving depression and anxiety , endocrine disorders including diabetes mellitus, male sexual dysfunction, cognitive dysfunctions etc. The conventionally used drugs to treat stress like benzodiazepine , anxiolytics , are capable of exerting effective antistress activity against acute models of stress only and have not proved effective against chronic stress induced adverse effects. Furthermore, these drugs are associated with many side effects and adverse reactions. At the same time many herbs reported in ancient literature have potent antistress activity and their utilities in current scenario need to be unveiled. Murraya Koenigii commonly called as curry leaf is widely used as culinary herb and herbal remedy for some common ailments. The whole plant is considered as tonic , stomachin and carminative Especially leaves are used to promote appetite, digestion and during dysentery, diarrhoea and to stop vomiting and to destroy pathogenic microorganisms¹³. It is reported to be useful in emaciation skin diseases, worm troubles ,necrosis and poisons. Roots are antiprotozoal and its juice is used to relieve pain associated with kidney^14. The stems are very popular for cleaning teeth and are said to strengthen the gums and teeth¹⁵. Murraya koenigii is known to be the richest source of carbazole alkaloids which are reported to possess various biological activities such as antitumor ,antioxidative, antimutagenic and antiinflammatory^16,17. etc. All parts of plants especially leaves are rich in carbazole alkaloids. Which include members with C13 skeleton murrayacin mukoeic acid,mukonone and mukonidine C18 skeleton including girinimbine, koenimbine, murrayacine, koenigine and koenigicine (koenidine) and C23 skeleton containing mahanimbine, mahanimbicine, isomahanimbicine, mahanine, mahanimbinine, murrayazoline, murrayazolinine, murrayazolidine, cyclomahanimbine, bicyclomahanimbicine Other carbazole bases includes mukoline, mukolidine (C13 from roots) mukonal (C13 from stem bark), mahanimboline (C23 from root and bark), isomurrayazoline (C23 from stem bark) ^18-24, The leaves contain a coumarin glycoside, scopolin²⁵and also essential oils containing sesquiterpenes and monoterpenes (ß caryophylline bgurjunene,b phellandrene etc)^26. These phytoconstituents contribute to various medicinal properties of Murraya koenigii .In the present studies efforts have been made to investigate antistress potential of Murraya Koenigii.

2. MATERIALS AND METHODS:

2.1. Plant material and extraction:

Murraya koenigii leaves were collected from the local market of Mumbai. The botanical authentification was done by the Department of Botany, Blatter Herbarium, St. John College Mumbai with voucher specimen (20537/SKW-2549) deposited in Blatter herbarium for the future reference.The fresh leaves were shade-dried, pulverized and passed through a 20-mesh sieve. The dried, coarsely powdered plant material was extracted with water methanol and 50% ethanol using Soxhlet apparatus. The solvents were evaporated under vacuum, which gave semisolid mass with respect to the dried powder. Oral suspensions containing 50mg/ml of the aqueous, methanolic and hydroalcoholic extract of Murray.Koenigii were prepared in 0.1% w/v Sodium CMC

2.2. Animals:

Albino wistar rats weighing 150–200 g of either sex were used for this study. The experimental animals were housed in polypropylene cages and maintained under standard conditions (12 h light and dark cycles, at 25⁰c ± 3⁰c and 35–60% humidity). Standard pelletized feed and tap water were provided ad libitum. The protocol for the study was approved by Institutional Animal Ethical Committee .Wistar rats of either sex were divided randomly into fourteen groups , each containing eight rats.

Group I : rats received 0.1% Na CMC in vehicle and not subjected to stress (Vehicle control group)

Group II : rats were treated with 0.1% Na CMC in vehicle and subjected to stress; (stress control group)

Group III : rats were treated with standard Diazepam (Calmpose 5 mg) (1mg/kg) i.p. and subjected to stress;

Group IV: rats were treated with standard Ashwagandha (Himalaya Ashvagandha 250mg) (100mg/kg) p.o. and subjected to stress .

Group V : rats were treated with Piracetam (Neurocetam 400 mg) (200 mg/kg) p.o. in saline and subjected to stress .

Group VI -VIII : rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp.p.o. and subjected to stress

Group IX -XI : rats were treated with MKHA extract at doses of 50,100 and 200 mg/kg resp.p.o. and subjected to stress

Group XII-XIV: rats were treated with MKM extract at doses of 50,100 and 200 mg/kg resp .p.o and subjected to stress.

The vehicle/ Standards/ extracts were administered daily.1 hr before subjecting to the immobilisation stress All groups except vehicle control group were subjected to immobilisation stress (2 hours/day)continuously for a period of 14 days.

2.3 Chronic restraint Stress:²⁷

Individual animal was restrained inside cylindrical plastic restrainer (19.5cm × 6.5cm ) daily for 2 hrs .The immobilisation procedure was performed between 10:00 and 12:00 h every day and was followed with the cognition studies between 12:00 and 16:00 h. on 1^st ,7^th and 14^th day of study .Blood was collected by retro orbital technique periodically on 1^st ,7^th and 14^th day of study, centrifuged at 4⁰c at 3000 rpm ×15 min and the serum was separated. The serum was used for estimation of various biochemical parameters such as corticosterone , glucose ,triglyceride and cholesterol. Animals were sacrificed at the end of the study period (14^th day) by cervical dislocation. Tissues like brain and adrenal glands were removed ,rinsed in isotonic saline and were weighed. Brain was isolated and immediately homogenized for estimation of catecholamines.

2.4 Estimations:

2.4.1.Estimation of Norepinephrine, Dopamine and 5-Hydroxy Tryptamine²⁸ :

To homogenizer tubes submerged in ice was added an ice-cold solution of acidified n-butanol. 10 ml of the acidified butanol per gram of tissue were used, and a motor driven Teflon pestle was used to homogenize the tissue..The homogenates were transferred to centrifuge tubes and tubes were centrifuged for 5 min at 3000 rpm .2.5ml aliquots of the supernatant fluid were transferred to tubes containing 1.6 ml of 0.1 N hydrochloric acid and 5 ml heptane. All tubes were shaken for 2 min and centrifuged for 5 min at 3000 rpm . The organic supernatant phase was aspirated and discarded, including the tissue disc at the interface of the sample tubes. Aqueous phase or acid extract was used for estimation of norepinephrine , dopamine and 5-HT using fluorospectrophotometric assays (Jasco, Japan).

2.4.2.Biochemical Estimations :

Alterations in the biochemical parameters were estimated using commercially available kits (Erba Diagnostics)

2.5. Cognitive assessment:

2.5.1 Transfer latency on Elevated Plus maze:²⁹

The elevated plus maze consisted of two opposite open arms (50 x 10 cm) crossed with two closed arms of the dimension with 40 cm high wall. The arms were connected with central square 10 x 10 cm to give the maze the shape of plus sign. The maze was elevated 50 cm above the floor and kept in dimly lit room. Rats were placed individually on one far end of an open arm and the time taken to enter one of the closed arms was recorded as the transfer latency. A day before giving stress and drug treatment the rat was given five trials at 10 min interval. The transfer latency was usually established by this time. Transfer latency (TL) was recorded on 1^st, 7^th and 14^th day of the study in order to assess the acquisition and retention of memory.

2.5.2 Step down inhibitory avoidance:³⁰.

The animals were trained for one way step down inhibitory task using a 50×25×25 cm plywood box with a Perspex wall front and a floor consisting of 1 mm bronze bars spaced 10 mm apart. The left end of the grid was covered with 5 cm high, 25 cm wide and 7.5 cm long wood platform³¹ A rat was placed on the platform and allowed to step down. Twenty-four hours later the animals were gently held by the body and lowered onto the platform , at which point a timer was activated to measure the latency to step down (i.e. placing all four paws on the grid) and on stepping down it received intrermittent foot shock (6mA) through the grid floor of 5 second duration until the animal climbed back on the platform. The rat was given three trials with inter trial interval of 30 min.for 3 days until the latency of the step down had stabilized prior to subjecting them to chronic restraint stress. In the test session on 1^st, 7^th and 13^th day of stress and treatment the test was repeated to record step down latency (SDL) to assess acquisition and retention of memory of learned task. During testing sessions 300 seconds ceiling was imposed, latencies >300 s were counted as 300 sec

Retention of memory for each animal was calculated in seconds (cut off point 300 s)

2.6. Statistical analysis:

All the values are expressed as mean ± SEM and data was analyzed by one-way ANOVA, using Graph pad INSTAT. The post hoc analysis was carried out by Dunnett’s test to estimate the significance of difference between groups.

Table no: 1 Effect of Murraya Koenigii extracts on serum corticosterone

Treatment groups	Corticosteron (ng/ml)
	Day1	Day7	Day14
	MEAN SEM	MEAN SEM	MEAN SEM
Vehicle control	11.83 ±1.08	10.19 ±1.31	9.70 ±0.56
Stress control	32.58^#±6.29	27.75^#±6.84	28.10^#±10.85
Diazepam 1mg/kg	27.60 ±2.80	20.65 ±4.25	20.18 ±2.49
Ashwagandha 100 mg/kg	20.94 ±4.15	17.71 ±4.94	13.43** ±2.90
MKAQ 50 mg/kg	31.93 ±4.54	22.79 ±2.31	21.49 ±2.13
MKAQ 100 mg/kg	26.95 ±3.63	21.50 ±2.90	19.86 ±2.20
MKAQ 200mg/kg	27.38 ±3.31	22.41 ±2.55	19.90 ±3.03
MKHA 50 mg/kg	28.40 ±4.54	21.59 ±3.37	20.99 ±2.41
MKHA 100 mg/kg	28.25 ±4.35	21.55 ±3.77	17.44* ±2.33
MKHA 200 mg/kg	27.75 ±3.37	19.05 ±2.67	16.48* ±2.05
MKM 50 mg/kg	31.50 ±4.72	22.20 ±2.75	20.16 ±2.37
MKM 100 mg/kg	27.23 ±3.23	19.25 ±1.74	16.51* ±1.39
MKM 200 mg/kg	27.51 ±4.40	17.11 ±2.45	15.39** ±2.01

n=6 to 8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group, #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

Table no: 2 Effect of Murraya Koenigii extracts on various biochemical parameters

Treatment groups	Glucose (mg/dl)			Cholesterol (mg/dl)			Triglycerides (mg/dl)
	Day 1	Day 7	Day 14	Day 1	Day 7	Day 14	Day 1	Day 7	Day 14
	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM
Vehicle control	72.38 ±3.98	70.38 ±3.06	70.125 ±1.97	100.58 ±1.88	98.74 ±2.19	101.48 ±2.96	63.13 ±3.02	64 ±2.99	64.45 ±2.48
Stress control	102.88^# ±1.69	96.27^# ±2.53	99.92# ±4.69	106.78 ±2.32	83.82^# ±3.04	80.20^# ±3.04	56.63 ±3.36	47.58^# ±3.15	46.52^# ±2.82
Diazepam 1mg/kg	100.05 ±2.68	98.84 ±2.38	96.44 ±2.12	105.69 ±2.82	85.33 ±3.48	86.60 ±2.56	57.69 ±2.32	50.91 ±2.56	51.35 ±1.84
Ashwagandha 100 mg/kg	99.86 ±1.89	83.19* ±2.49	69.74** ±3.16	104.30 ±2.29	97.68* ±3.89	97.75** ±2.84	59.73 ±2.93	61.51* ±2.84	58.64** ±1.97
MKAQ 50 mg/kg	109.81 ±3.35	101.86 ±2.35	104.63 ±3.50	101.11 ±3.53	82.86 ±3.12	82.21 ±2.75	48.03 ±2.64	46.23 ±2.84	47.80 ±1.72
MKAQ 100 mg/kg	105.94 ±3.47	90.73 ±3.63	85.70* ±3.27	105.26 ±2.34	86.93 ±2.66	86.95 ±2.36	58.43 ±1.41	50.56 ±1.31	52.71 ±2.08
MKAQ 200mg/kg	99.30 ±1.92	91.36 ±2.15	80.59** ±3.69	105.54 ±2.39	84.09 ±2.91	87.41 ±1.85	53.59 ±2.87	56.86 ±1.82	55.16* ±1.74
MKHA 50 mg/kg	105.94 ±2.39	104.83 ±1.42	103.86 ±2.33	105.44 ±2.46	85.00 ±3.18	88.05 ±3.17	56.43 ±2.85	48.83 ±1.61	49.98 ±1.94
MKHA 100 mg/kg	101.19 ±1.96	91.67 ±3.90	86.20* ±2.99	109.06 ±2.85	87.75 ±3.00	92.18* ±3.37	57.33 ±2.24	50.70 ±2.03	55.41* ±2.29
MKHA 200 mg/kg	102.41 ±2.33	91.42 ±4.36	82.01** ±3.47	103.76 ±2.25	95.68 ±2.34	92.79* ±2.04	57.98 ±2.85	54.75 ±1.64	56.12* ±2.15
MKM 50 mg/kg	105.06 ±2.21	94.21 ±3.29	99.93 ±3.12	105.14 ±2.13	89.94 ±3.14	86.16 ±3.30	49.73 ±2.46	50.93 ±2.37	53.53 ±1.27
MKM 100 mg/kg	100.23 ±2.62	86.14 ±2.94	84.59** ±2.69	99.72 ±3.19	91.68 ±3.73	91.69* ±3.51	50.55 ±2.43	53.34 ±2.88	56.59* ±2.00
MKM 200 mg/kg	101.55 ±3.47	82.31* ±3.47	72.80** ±2.74	102.23 ±3.02	91.39 ±3.24	97.06** ±2.31	55.84 ±2.84	60.33* ±2.01	60.08** ±2.84

n=6 to 8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group, #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

3. RESULTS:

3.1. Biochemical investigations:

(a) CRS induced marked increase in plasma corticosterone level as compared to vehicle control group which was inhibited significantly (P<0.01) by MKM (200 mg/kg) on 14^th day (45.24% ) was comparable to the effect produced by standard drug AS (100mg/kg) (52.22 %) on 14^th day whereas moderately significant effect (P<0.05) was observed at dose 100 mg/kg (41.23 %). MKHA (100 and 200 mg/kg) was found to be effective (P<0.05) on 14^th day (37.94% and 41.37% resp.) whereas effect produced by MKAQ (200mg/kg) on 14^th day of study was found to be statistically insignificant. Similarly inhibitions of stress induced elevated levels of corticosterone observed at 50mg/kg of dose for MKM, MKHA and MKAQ were not found to be statistically significant.(P>0.05) (Table no1 ).

(b)CRS adversely affected blood glucose concentration. The stress-induced hyperglycaemia was attenuated significantly (P<0.01) by MKM 200mg/kg on 14^th day (27.10%) and was comparable to that produced by standard AS (30.57%) whereas its effect on 7^th day (15.07%) was found to be moderately significant. At dose 100 mg/kg significant (P<0.01) reduction in blood glucose levels (16.93%) was observed only on 14^th day of study. MKHA (100 mg/kg) (13.7%) and. MKAQ 200mg/kg (19.34%) showed moderately significant (P<0.05) restoration in blood glucose levels on 14^th day of study as compared to stress control group.Diazepam treatment failed to produce any significant effect on stress induced hyperglycemia (Table no:2 )

(c)CRS induced fall in levels of serum Triglycerides on 7^th and 14^th day of study (25.65% and 27.82% respectively) in stress control group as compared to vehicle control group which were prevented by MKM (200mg/kg) on both 7^th (26.77%(P<0.05)) and14^th day (29.14% (P<0.01)) of study whereas MKM (100mg/kg) produced the effect (26.77% (P<0.05)) only on 14^th day of study. Effects observed with MKM were comparable with that produced by standard AS on 7^th (29.27% (P<0.05)) and 14^th (26.05% (P<0.01))day of study Restoration in levels of plasma triglycerides were observed with MKHA (100 and 200mg/kg) (19.12% and 29.01% resp. (P<0.05)) and MKAQ (200mg/kg) (18.58%(P<0.05)) on 14^th day of treatment as compared to stress control group. (Table no: 2)

(d)Cholesterol levels were found to be increased on 1^st day of restraint stress non significantly (P>0.05) to an extent of 6.2% where as on 7^th and 14^th day of stress, levels were found to be declined significantly (P<0.01) to an extent of -15.11% and -20.96% resp. in stress controlled group as compared to vehicle control group .On treatment with MKM ( 200 mg/kg) stress induced decrease in levels of cholesterol was significantly (P<0.01) prevented on 14^th day (-21.51%) and was observed to be similar to that produced by standard AS (-21.88%). On treatment with MKHA 100 mg/kg (-14.93%) and 200 mg/kg (-15.69%) moderately significant (P<0.05) effect was observed only on 14^th day of treatment whereas treatment with MKAQ failed to produce statistically significant restoration in levels of Cholesterol during CRS. (Table no:2 )

3.2Weight of Adrenal glands:

Stress induced hypermegaly of adrenal glands was observed in animals treated with CRS to an extent of 72.52% which was found to be decreased to an extent of 28.74% and 37.19% (P<0.01) on treatment with MKM (100 and 200 mg/kg resp). On treatment with MKHA (200 mg/kg) an effect of 34.06% (P<0.01).and with MKAQ (200 mg/kg) an effect of 12.95% was observed .Standard AS showed significant effect of 43.45% (P<0.01) in contrast to stress control group. (Table no:3 )

Table no:3 Effect of Murraya Koenigii Extracts on adrenal glands weight

Treatment groups	Mean wet weight of adrenal glands (gm/100 gm body weight)
Vehicle control	0.011 ±0.001
Stress control	0.020^#±0.002
Diazepam 1mg/kg	0.019 ±0.002
Ashwagandha 100 mg/kg	0.011** ±0.001
MKAQ50 mg/kg	0.020 ±0.002
MKAQ 100 mg/kg	0.019 ±0.002
MKAQ 200mg/kg	0.017 ±0.001
MKHA 50 mg/kg	0.019 ±0.002
MKHA 100 mg/kg	0.015 ±0.001
MKHA 200 mg/kg	0.013** ±0.001
MKM 50 mg/kg	0.017 ±0.002
MKM 100 mg/kg	0.014* ±0.001
MKM 200 mg/kg	0.012** ±0.001

n=6 to8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group, #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

3.2 Brain Bioamine levels:

Restraining of the animals for 120 min every day for 14 days induced significant (P<0.001) decrease in brain levels of Nor epinephrine (65.65 %) and elevation in 5HT (68.45 %) and dopamine (111.55 %) levels in stress control group as compared to vehicle control (unstressed) group.

MKM (100 and 200 mg/kg) prevented fall in levels of norepinephrine to an extent of 124.39% and 135.9% resp which was moderately significant (P<0.05).Prevention in stress induced decrease in levels of norepinephrine by MKHA (200 mg/kg) was observed to be 131.39% (P<0.05)and with that of MKAQ treatment was not found to be statistically significant (P>0.05) as compared to stress control group. Standard AS prevented fall in levels of nor epinephrine to an extent of 134.17% (P<0.05) whereas effect produced by Diazepam was not statistically significant (P>0.05).

CRS induced rise in levels of 5HT in rat brains (68.45%) which was prevented by treatment with MKM (200 mg/kg) to an extent of 37.01%.and with MKHA (200mg/kg) to an extent of 33.16% and were observed to be moderately significant(P<0.05). Effect shown by MKAQ treatment on 5HT levels was not statistically significant.

Treatment with MKM (100 and200 mg/kg) prevented stress induced rise in levels of brain dopamine to extent of 40.66% and 38.58% resp. whereas treatment with MKHA (100 and 200mg/kg) produced an effect of 27.52% and 40.72% resp. during CRS which was though not statistically significant but was able to restore dopamine levels near to the levels of dopamine observed in vehicle control group (Unstressed group) Standard AS exerted the effect to an extent of 39.16% in preventing stress induced rise in levels of dopamine. Effect of standard Diazepam on levels of bioamines during CRS was not observed to be statistically significant (P>0.05). (Table no:4 )

Table no.4 Effect of Murraya Koenigii Extracts on Levels of Bio amines in rat brain

Treatment groups	Nor epinephrine μg/gm wet brain	5-Hydroxy Tryptamine μg/gm wet brain	Dopamine μg/gm wet brain
	Mean ±SEM	Mean ±SEM	Mean ±SEM
Vehicle control	0.47 ±0.04	0.31 ±0.05	1.34 ±0.17
Stress control	0.16 ±0.04	0.52 ±0.03	2.84 ±0.35
Diazepam 1mg/kg	0.20 ±0.04	0.46 ±0.04	2.22 ±0.38
Ashwagandha100mg/kg	0.38* ±0.06	0.34** ±0.03	1.73 ±0.27
MKAQ 50 mg/kg	0.20 ±0.03	0.54 ±0.02	2.68 ±0.33
MKAQ 100 mg/kg	0.20 ±0.03	0.47 ±0.03	2.30 ±0.36
MKAQ 200mg/kg	0.24 ±0.03	0.48 ±0.04	2.46 ±0.19
MKHA 50 mg/kg	0.24 ±0.06	0.44 ±0.02	2.45 ±0.34
MKHA 100 mg/kg	0.32 ±0.06	0.40 ±0.05	2.06 ±0.29
MKHA 200 mg/kg	0.37* ±0.05	0.35* ±0.03	1.68 ±0.26
MKM 50 mg/kg	0.18 ±0.04	0.53 ±0.06	2.44 ±0.47
MKM 100 mg/kg	0.36* ±0.06	0.39 ±0.04	1.74 ±0.27
MKM 200 mg/kg	0.38* ±0.04	0.33* ±0.04	1.68 ±0.21

n=6 to 8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group, #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

3.3. Effect on Cognitive functions:

CRS significantly and adversely affected retention of memory as observed with significant (P<0.01) increase in the transfer latency (TL) on 7^th (44.74%) and 14^th (57.75%) day and significant (P<0.01) decrease in step down latency (SDL) on 7^th day (48.68%) and moderately significant (P<0.05) effect on 14^th day (54.69%) in contrast to vehicle control group. Standard Piracetam significantly (P< 0.01)) and effectively prevented these stress induced alterations in TL (-11.2%,-9.2%) and SDL (207.59% 244.09%) on 7^th and 14^th day of treatment resp.MKM at doses 100 and 200 mg/kg could significantly (P<0.01) prevent increase in TL in EPM on 14^th day (32.21% and 32.84% resp.) and produced moderately significant effect on 7^th day (27.32%) of treatment. Treatment with MKHA (200mg/kg) significantly (P<0.01) produced the effect by preventing stress induced increase in TL on both 7^th (31.65%) and 14^th (35.66%) day of treatment. Treatment with MKAQ produced moderately significant effect only on 14^th day of study (29.07%) as compared to stress control group. (Table no:5 )

Table no.5 Effect of Murraya Koenigii Extracts on Transfer latency in Elevated plus maze

Treatment groups	Transfer latency (TL) (seconds)
	Day1	Day7	Day14
	Mean ±SEM	Mean ±SEM	Mean ±SEM
Vehicle control	15.88 ±2.59	23.38 ±2.45	25.25 ±2.15
Stress control	27.13^# ±1.98	33.83 ±2.59	39.83^# ±2.17
Piracetam 200mg/kg	19.00** ±2.01	19.63** ±2.06	17.75** ±1.30
Diazepam 1mg/kg	26.88 ±2.28	37.63 ±1.46	43.50 ±2.44
Ashwagandha 100mg/kg	20.75 ±2.42	28.75 ±2.26	29.88 ±1.82
MKAQ 50 mg/kg	25.00 ±2.43	30.25 ±2.13	36.14 ±2.90
MKAQ 100 mg/kg	25.25 ±3.08	30.75 ±3.13	38.00 ±3.30
MKAQ 200 mg/kg	21.75 ±2.91	26.00 ±2.42	28.25* ±1.50
MKHA 50 mg/kg	25.75 ±2.54	32.75 ±1.79	37.00 ±3.01
MKHA 100 mg/kg	24.00 ±2.01	35.25 ±2.99	34.38 ±2.51
MKHA 200 mg/kg	25.88 ±2.40	23.13** ±1.76	25.63** ±2.08
MKM 50 mg/kg	30.50 ±2.25	31.57 ±1.66	34.29 ±2.71
MKM 100 mg/kg	27.75 ±3.40	27.38 ±2.25	27.00** ±2.31
MKM 200 mg/kg	25.38 ±2.65	24.25* ±1.64	26.75** ±1.61

n=6 to 8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group, #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

MKM (100 and 200mg/kg) exerted moderately significant effect on SDL on 14^th day (100.67% and 110.41% resp.) whereas MKM (200mg/kg) exhibited moderately significant effect (97.23%) on 7^th day of treatment. Treatment with MKHA (100 and 200mg/kg) showed moderately significant effect (P<0.05) on both 7^th day (85.36% and 88.66% resp.) and 14^th day (98.68% and 103.92% resp.) of study whereas MKAQ failed to produce any statistically significant effect as compared to stress control group. Effects demonstrated by MKM (200 mg/kg) on TL and SDL were significantly higher than that produced by other extracts on 14^th day of treatment. (Table no:6 )Thus from the above results MKM (200 mg/kg) appears to be comparatively the most effective extract in normalising most of the stress induced physiological perturbations.

Table no: 6 Effect of Murraya koenigii on step down latency

Treatment groups	Step down Latency (SDL) (Seconds)
	Day1	Day7	Day14
	MEAN ±SEM	MEAN ±SEM	MEAN ±SEM
Vehicle control	122.25 ±11.79	102.63 ±8.27	93.50 ±8.48
Stress control	96.63^$ ±4.04	52.67^# ±7.15	42.36^# ±5.51
Piracetam 200 mg/kg	131.75 ±8.04	162.00** ±12.18	145.75** ±9.71
Diazepam 1mg/kg	105.75 ±5.84	77.13 ±9.74	57.88 ±7.66
Ashwagandha 100mg/kg	137.13 ±11.72	104.63* ±15.42	83.25* ±13.20
MKAQ 50mg/kg	125.38 ±8.88	90.00 ±11.89	48.67 ±13.34
MKAQ 100mg/kg	122.00 ±8.53	91.50 ±6.67	60.63 ±6.49
MKAQ 200 mg/kg	120.13 ±9.45	91.63 ±10.71	65.88 ±7.72
MKHA 50mg/kg	122.00 ±7.83	90.75 ±7.90	67.13 ±9.41
MKHA 100mg/kg	121.00 ±12.84	97.63* ±14.50	84.13* ±12.11
MKHA 200 mg/kg	129.63 ±13.17	99.38* ±13.20	86.38* ±14.63
MKM 50mg/kg	121.13 ±20.34	83.57 ±21.10	53.57 ±16.75
MKM 100mg/kg	125.75 ±7.37	92.25 ±10.55	85.00* ±8.75
MKM 200 mg/kg	124.88 ±11.21	103.88* ±13.34	89.13* ±11.26

n=6 to 8 the values are expressed in Mean ± SEM ,**=p<0.01,*=p<0.05 when compared with stress control group. #=p<0.01,$<0.05 when compared with vehicle control group (one-way ANOVA followed by Dunnett’s t-test or Student’s unpaired t-test)

4. DISCUSSION:

Stress in rats brings about transient activation of the HPA axis, as measured by increased adrenal gland weight with subsequent increase in plasma corticosterone level and other correlates of adrenal activation which prepares the organism for threatened homeostasis³². The important biochemical changes in plasma under stressful conditions, i.e. elevated corticosterone is necessary to maintain the energy balance which include increased plasma glucose, and decreased triglyceride and cholesterol levels³³ Under stressful condition adrenal cortex secrets cortisol in man and corticosterone in rats. Hypersecretion of cortisol helps in maintainance of internal homeostasis through the process of gluconeogenesis and lipogenesis³⁴ In present study stress induced significant hyperglycaemia was inhibited by treatment with extracts of Murraya koenigii. Diabetes mellitus is a well accepted consequence of continuous stress, indicating the close interrelations between stress and the endocrine and autonomic nervous systems³³³⁵ In accordance to literature survey Murraya koenigii has been reported to possess hypoglycemic effect^{36, 37} and in present work it is also found to be effective in normalising stress-induced perturbation of glucose homeostasis. The stress raises utilisation of serum cholesterol resulting in increased liberation of catecholamines and corticosteroids through enhanced activity of hypothalamohypophyseal axis³⁸ In present study restraint stress induced reduction in levels of plasma cholesterol as compared to vehicle control group (unstressed) but this effect of stress on levels of cholesterol was ameliorated following treatment with leaf extracts of MK. Effect of stress on triglyceride levels is found to be variable probably due to mobilisation of lipids from adipose tissues by catecholamines released in high concentration during stress. Results of present study showed decline in levels of triglycerides in stress control group but on treatment with MK extracts MKM (200 mg/kg) was observed to be the most effective in preventing this stress induced decrease in levels of triglycerides which might be due to suppression of stress induced lipolysis .Probably an effective MK extracts are able to promote assimilation of glucose in tissues as an immediate source of energy as a result breakdown of lipids as an alternative source of energy was reduced which resulted in prevention of decline in levels of triglycerides. These observed effects of extracts on biochemical parameters are interdependent and might be due to suppressant effect of MK on hyperactivity of adrenal cortex. Likewise, MK extracts inhibited the significant increase in plasma corticosterone levels (MKM was observed to be the most effective) induced by restraint stress. This normalizing effect on plasma corticosterone is one of the possible reasons for its anti stress properties.

This observed anti stress effect was further investigated to understand the mechanism by measuring bioamines levels in brain . Various workers have given different opinions about changes in the levels of brain monoamines, This may be due to the nature of stressor , duration of stressor and the animal used in experiments. In our study immobilization of 120 min for 14 days continuously resulted in significant decrease in levels of Noradrenalin and increase in 5-HT and Dopamine levels in brain. Stressful conditions activate monoaminergic system leading to an increase in the turnover of Noradrenalin in cortex and hippocampus regions of brain³⁹ It has been suggested that dopamine levels in brain elevate as a compensatory mechanism and as a precursor for synthesis of more norepinephrine to cope with increased demand⁴⁰ where as 5HT levels in brain increases in response to physical and psychological stress due to immobilisation in restraint stress⁴¹.All three extracts methanoloic ,hydroalcoholic and aqueous extract of MK were effective at variable extents in resisting these perturbations in levels of NA ,5-HT and Dopamine in rat brain. but the most significant and effective one observed to be was methanolic extract.These normalising effects of MK extracts on bioamine levels probe in to the probable mechanism involved in preventing stress induced biochemical alterations on treatment.

During stressful conditions, changes in monoamines (NA, DA and 5-HT) are well associated with transient behavioural aberrations in memory learning and other mood disorders. Experimental stress was reported to have adverse effects on the memory engram in rats. The learning acquisition was minimally affected, the major action being disruption of retention of learned tasks⁴². MK was reported earlier to have significant nootropic activity⁴³ but its effect on stress induced amnesia is not established. In this study when tested for retention of learned task in elevated plus maze after exposing to restraint stress, groups treated with MK showed decrease in Transfer latency period on 7^th and 14^th day of test and similarly in step down inhibitory avoidance test Step down latency period was found to be increased suggesting the ability of extracts to avoid detrimental effects of stress on memory. The exact mechanism for these observations cannot be explained with our present data, however the role of corticosterone can be hypothesized, as such effects of stress are inhibited by CRF antagonist and adrenalectomy suggesting the possible mediation of corticosterone⁴⁴.The various physiological changes seen in response to stress are primarily due to increased hypothalamo pituitary action which in turn induces activation of pituitary adrenal system. Adrenaline stimulates β2 receptors on the pituitary gland causing greater release of ACTH which in turn can stimulate the adrenal medulla as well as cortex leading to adrenomegaly. In this study in treatment groups significant restoration of adrenal gland weight was observed which shows potential role of MK in attenuating the activation of HPA axis. The carbazole alkaoloids and terpenes found to be present in MK may be probably contributing to the antistress potential of plant and further study needs to be carried out but from the present data one can conclude that Murraya koenigii may provide a protection against stress probably by preventing biochemical and humoral perturbations during stress and their adverse implications on body physiology and thus can be a safe alternative antistress agent for therapy of stress related disorders.

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Received on 13.04.2011

Accepted on 29.06.2011

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Research J. Pharmacology and Pharmacodynamics. 3(4): July –August, 2011, 184-191