FORM 2
THE PATENTS ACT, 1970 (39 OF 1970) & THE PATENTS RULES, 2003 PROVISIONAL/COMPLETE SPECIFICATION (See section 10 and rule 13)
i TITLE OF THE INVENTION. : ISOLATION OF CARBAZOLE
ALKALOIDS FROM MURRAYA KOENIGII LEAVES
9. APPLICANTS
Name Nationality Address
1. Mrs.MRINAL MANOJ SANAYE Indian 404,Nebula -C,
Cosmos Paradise, Devdaya Nagar, Thane (west), Thane-400606
2 Dr. MADHUSUDAN NATVARLAL SARAF Indian
103,Jupiter II, Navkiran Marg Off four bunglows Andheri (West), Mumbai 400053
3. PREAMBLE TO THE DESCRIPTION
COMPLETE: The following specification particularly describes the invention and the manner in which it is to be performed
4.DESCRD?TION:( Description shall start from the next page )
5. CLAIMS (not applicable for provisional specification. Claims should start with the preamble —
"I/We claim" on separate page)
6. DATE AND SIGNATURE (to be given at the end of last page of specification)
7. ABSTRACT OF THE INVENTION:

Complete patent specification
ISOLATION OF CARBAZOLE ALKALOIDS FROM MURRAYA KOENIGII LEAVES
FIELD OF INVENTION
The present invention deals with the isolation of carbazole alkoloids from the leaves of Murraya koenigii especially the Mahanimbine and the use of extract as an anti-stress agent.
BACKGROUND OF THE INVENTION :
Murraya koenigii, an aromatic leaf often used in Indian cuisine. The leaves are highly valued as seasoning in southern, west-coast Indian cooking, and Sri Lankan cooking-especially in curries. Murraya Koenigii has been claimed as tonic and is highly valued as folk medicine and functional food
In scientific literature Murraya Koenigii is known to be a rich source of carbazole alkaloids (3) and is reported to possess various biological activities such as anti tumor ,anti-oxidative, hypoglycemic hypolipidemic and anti-inflammatory etc (1,2,3,4,5,6)
The bioassay guided fractionation of the acetone extract of the fresh leaves of Murraya koenigii resulted in the isolation of three bioactive carbazole alkaloids, mahanimbine (1), murrayanol (2), and mahanine(3) , as confirmed from their 1H and 13C NMR spectral data. Compound 2 showed an IC50 of 109 μg/mL against hPGHS-1 and an IC50 of 218 ug/mL against hPGHS-2 in anti-inflammatory assays, while compound 1 displayed antioxidant activity at 33.1 ug/mL. All three compounds were mosquitocidal and antimicrobial and exhibited topoisomerase I and II inhibition activities.( Russel S. Ramsewak, Muraleedharan G. Nair, Gale M. Strasburg, David L. DeWitt, and John L. Nitiss, J. Agric. Food Chem., 1999, 47 (2), pp 444-447)

Mahanimbine and koenigine, were the two carbazole alkaloids, isolated from the leaves of M. koenigii which showed antioxidant activity. Koenigine also showed a high degree of radical-scavenging properties (Julie Banerji et al J. Chem. Pharm. Res., 2010, 2 (2): 286-299)
A new dimeric carbazole alkaloid, 8,10'-[3,3',ll,11'-tetrahydro-9,9'-dihydroxy-3,3',5,8'-tetramethyl-3,3'-bis(4-methyl-3-pentenyl)]bipyrano[3,2-a]carbazole,was isolated from the CH2C12 extract of Murraya koenigii together with six known carbazole alkaloids, koenimbine , O-methylmurrayamine A , O-methylmahanine , isomahanine , bismahanine , and bispyrayafoline .. The anti-oxidative properties of 12 carbazole alkaloids isolated from leaves of M. koenigii were evaluated on the basis of the oil stability index together with their radical scavenging ability against l,l-diphenyl-2-picrylhydrazyl (DPPH) radical. .( Yukari Tachibana,Hiroe Kikuzaki, Nordin Hj. Lajis, and Nobuji Nakatani;( /. Agric. Food Chem., 2003,51 (22), pp 6461-6467)
Studies have proved the antidiabetic effect of the Murraya koenign extract In an animal study in the diabetic rats, it was observed that the elevated fasting blood sugar, triglycerides, low density lipoprotein, very low density lipoprotein levels were reduced and high density lipoprotein level was increased by mahanimbine at a dose of 50 and 100 mg/kg (i.p). In addition, mahanimbine showed appreciable alpha amylase inhibitory effect and weak alpha glucosidase inhibitory effects when compared with acarbose. It was concluded that mahanimbine possess anti-hyperglycemic and anti-lipidemic effects. Thus results suggesting mahanimbine has beneficial effect in the management of diabetes associated with abnormal lipid profile and related cardiovascular complications^ B. Dineshkumar, Analava Mitra, Manjunatha Mahadevappa JntJ.Phytomedicine ,Vol.2,no.l,2010;p22-30: Antidiabetic and hypolipidemic effects of mahanimbine (carbazole alkaloid) from murraya koenigii (rutaceae) leaves).
Though the multiple benefits and medicinal uses of the extract (or the carbazole alkoloids like Mahanimbine )of leaves of Murraya koenigii are known-its effect as an anti-stress agent is largely unknown. Excess stress, generated by today's lifestyle can

manifest itself in a variety of emotional, behavioural, and even physical symptoms, and the symptoms of stress vary enormously among different individuals. Common somatic (physical) symptoms often reported by those experiencing excess stress include sleep disturbances, muscle tension, muscle aches, headache, gastrointestinal disturbances, and fatigue. Emotional and behavioural symptoms that can accompany excess stress include nervousness, anxiety, changes in eating habits including overeating, loss of enthusiasm or energy, and mood changes, like irritability and depression with increased vulnerability to diseases, impaired growth and reproductive function, osteoporosis, diabetes, cardiovascular diseases dementia and reduced life expectancy .
Hence there is a definite need for an effective anti stress agent which augment resistance to stress, and increase concentration, performance and endurance during fatigue and resist the untoward effects of stress on human body .There is no firmly established treatment recommendations for this condition in the conventional allopathic medicine and the available therapies in modern medicine are limited ,Thus the exploration of potential alternative therapies from traditional medicine is prerequisite to overcome stress.
Different methods have been tried for isolating various carbazole alkaloids from the leaves of Murraya koenigii. Ramsewak RS et al used bioassay guided fractionation of the acetone extract of the fresh leaves of Murraya koenigii This resulted in the isolation of three bioactive carbazole alkaloids, mahanimbine (1), murrayanol (2), and mahanine (3), as confirmed from their (1)H and (13)C NMR spectral data.( Agric Food Cheml999 Feb;47(2):444-7 )
In another study a dark green ethanol extract was obtained from 270 g of leaves and an aliquot (5 g) was subjected to silica gel flush column chromatography. Six fractions were obtained by gradient elution using a hexane-ethyl acetate solvent system.( Thilahgavani Nagappan et al; Molecules 2011,16, 9651-9664,)
Kumar NS isolated the carbazole alkaloid, mahanimbine [3, 5-dimethyl-3-(4-methyIpent-3-enyI)-llH-pyrano [5, 6-a] carbazole], from the petroleum ether extract of the leaves of Murraya koenigii.( Phytother Res 2010 Apr;24(4):629-31 )

In another method reported in International Journal of Phytomedicine 2,2010,22-30 following method was used for isolation of mahanimbine :The dried plant powder of Murraya koenigii leaves were extracted with petroleum ether(60-80°C) in a Soxhlet apparatus for 72 h. at room temperature. The total extract was concentrated under reduced pressure and kept at room temperature. A greenish solid (3.6%) was separated out. This was dissolved in petroleum ether (60-80°C) and chromatographed using silica gel (60-120 mesh) column and eluted successively with petroleum ether and chloroform mixture. The fractions obtained with 50% petroleum ether (60-80°C) in chloroform afforded compound-I (mahanimbine). The compound-I was subjected to preparative TLC gave pure mahanimbine (0.4%). (B. Dineshkumar et al International Journal of Phytomedicine 2 (2010) 22-30)
OBJECT OF THE INVENTION:
The main object of the present study was to develop an efficient method of extraction of Carbazole alkoloids including mahanimbine from the leaves of Murraya koenigii.
Another object was to develop an extraction method which not only gives better yield of carbazole alkoloids but also is simple and cost effective.
Yet another object of the present invention is to isolate a carbazole alkaloid with the anti-stress activity from the extract ofMurraya koenigii leaves.
DETAILED DESCRIPTION OF THE INVENTION
Method of extraction of carbazole alkaloid & Mahanimbine from Murraya koenigii leaves
The Murraya koenigii leaves were collected from local market. The leaves were air dried and powdered. The dried powder (0.3 KG) of Murraya koenigii passed through 40# was extracted with methanol. The extract (20% w/w) was dissolved in methanol acidified

with gl. Acetic acid and the solvent was evaporated under reduced pressure. To the resultant concentrated semisolid mass water was added and filtered. The filtrate was collected pH was adjusted to acidic and subjected for liquid liquid extraction with Methyl Isobutane ketone (MIBK) solvent. On separation of two phases aqueous phase was discarded whereas Methyl Isobutane ketone (MIBK) phase was collected and concentrated as an alkaloid fraction
Total 14 gm of MIBK fraction was chromatographed where at a time 1 gm of Methyl Isobutane ketone (MTBK) fraction was chromatographed on column (100 cm x 1.3 cm) of silica gel (60/120 #) and Chloroform elutes (5ml) were collected and the progress of chromatogram was followed by TLC . Fractions 16- 27 (Eluent : chloroform) collected during each chromatogram were combined (840 ml) and the solvent was distilled off .The residue on crystallization from hexane gave Mahanimbine (0.165g) thus producing 0.055% w/w of yield. Then the structure of Mahanimbine was characterized by noting down IR spectrum UV max values. Structural elucidation was done by recording Proton NMR and 13C NMR and Mass spectrum. From the results of these data the structure of isolated carbazole alkaloid was confirmed as Mahanimbine. (C23H25NO).
Common method reported in literature:
Joshi et al had reported the method of isolation of Mahanimbine in journal Tetrahedron According to this method the dried powdered leaves collected in the local market (9.5KG) were extracted with hexane (301x3) at room temperature. The extracts were combined and the solvent was evaporated under reduced pressure. The waxy residue (200g) was dissolved in benzene and chromatographed on Slica gel (2kg, .2-.5 mm ) in benzene. The eluted fractions (100ml) were collected and the progress of the chromatogram followed by TLC.Fractions 19-25 (eluent : benxene) were combined and the solvent was distilled off. The residue on crystallization from hexane gave mahanimbine (4 g) thus yielding 0.04% w/w of Mahanimbine. (Tetrahedron. 1970 Vol. 26: 1475-1482)

Modifications made for increasing the yield of alkoloids /Mahanimbine:
l).Greater yield: In reported method yield obtained is 0.04%w/w where as by our method the yield obtained is(0.055%w/w).
2).Bioactivity guided isolation of Mahanimbine: In already reported method no previous work on pharmacological activity of hexane extract was done before going for isolation of Mahanimbine whereas in our method methanol extract was previously tested for biological activity and when found effective was processed for isolation.
3 )Alkaloids being weakly basic in nature were selectively separated from extract as a fraction of carbazole alkaloids by forming a salt with gl acetic acid
4). Methyl isobutyl ketone (MIBK) is a non polar solvent having high stability with acidic solvent thus was used for liquid- liquid extraction to facilitate maximum isolation and effective separation of highly nonpolar carbazole alkaloid from alkaloid fraction leaving behind unwanted constituents in aqueous media.
5) This selective separation of only alkaloid fraction from the extract and then isolating a desired alkaloid from the fraction might be responsible for giving greater yield of an alkaloid in contrast to the application of whole crude hexane extract for chromatographic Separation of alkaloid
Testing of methanolic extract of Murraya koenigii for preventing stress induced rise in serum cortisone levels:
Extract of Murraya koenigii was found to be significantly preventing the stress induced rise in levels of serum corticosterone and thus even preventing stress induced rise in serum glucose levels in wistar rats subjected to restraint stress. It was also observed to be reducing stress induced increased turn-over of nor epinephrine in brain. The extract was also observed to possessing significant anti fatigue activity by preserving glycogen stores in muscle and liver tissues in mice thus prolonging their ability to continue doing swimming exercise for a longer period when tested on weight loaded forced swim test.

The extract was also found to be significantly effective in preventing depression induced by chronic stress in rats. When tested for its effect on cognition and memory extract was significantly effective in retaining of memory of learned task. The extract was significantly effective in avoiding oxidative stress induced damage by promoting activity of anti oxidant defense mechanism or may be due to inherent antioxidant property of the plant thus scavenging free radicals formed in oxidative stress. Thus extract was found to be significantly effective in prevention of physiological perturbations due to stress. Mahanimbine was also found to be possessing significant antioxidant effect In vitro when tested for DPPH radical scavenging activity and Lipid peroxidation
OTHER EFFECTS OF THE EXTRACTS OF MURRAYA: Stress Model(RS)(554)
Drug treatments : Vehicle (0.1% equi volume) Diazepam (lmg/kg p.o) (Calmpose 5mg Ranbaxy Lab. LTD), Ashwagandha (lOOmg/kg p.o)(Ashvagandha Himalaya drugs ) Piracetam (200 mg/kg p.o.) () and extracts MKAQ (50,100 and 200mg/kg p.o;Murraya Koengii -aqueous ext,) ,MKHA (50,100 and 200mg/kg p.o Murraya Koengii Hydro alcoholic ext.) and MKM (50,100 and 200mg/kg p.o Murraya Koengii methanolic Ext) were administered to the respective groups successively for a period of 14 days one hour prior to restraint stress.
Method:
Animals were trained to establish transfer latency on EPM and step down latency before subjecting them to restraint stress. All groups except vehicle control group were subjected to immobilization stress for 2 hours/day successively for a period of 14 days. lhour after the respective treatments.
The restraint stress was applied by placing each rat in a plastic restrainer. The Plastic restraint tubes were equipped with air holes and an adjustable end plate, which helped to account for differences in body weight and length and secured the rat within the tube. Once the rat was inserted in their respective plastic tube the end plate was lowered down to compress the animal in restrainer and was screwed tightly to immobilize the animal. It's tail was pasted with adhesive tape to further restrict the change in posture. Then the cohort was placed in a temperature control and sound proofroom separate from the main animal colony. Individual animal was restrained inside cylindrical plastic restrainer

(19.5cm x 6.5 cm ) daily for 2 hrs .The immobilization procedure was followed with the cognition studies on 1st ,7th and 14th day of study .Blood was collected from retro orbital plexuses immediately at the end of restraining procedure periodically on 1st ,7th and 14th day of study, centrifuged at 4°c at 3000 rpm x15 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 (14th 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 , Nor epinephrine, 5HT and Dopamine,
COGNITIVE ASSESSMENT: A. Elevated Plus maze [555].
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. Rat was 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 (TL). 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 1st, 7lh and 14th day of the study in order to assess the retention of memory of learned task B.Step down inhibitory avoidance [556]
The animals were trained for one way step down inhibitory task using a 50x25x25 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 with it's all four limbs 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 intermittent 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 1st, 7th and 14th 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 ceiiing 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) BIOCHEMICAL ESTIMATIONS :
> Alterations in the following biochemical parameters were estimated using
commercially available kits (Erba Diagnostics)
> Serum glucose levels
> Serum triglyceride levels
> Serum cholesterol levels
> Estimation of serum Corticosterone:was performed in Harmone lab, Mumbai by
radio-immuno assay method
ESTIMATION OF CATECHOLAMINES:
At the end of the study after blood withdrawl and cognitive studies animal were killed humanely by cervical dislocation without giving anaesthesia. Brains were isolated cleaned with isotonic saline solution and were immediately homogenized for catecholamine estimations. HISTOPATHOLOGICAL ESTIMATION I
Immediately after decapitation the adrenal glands were weighed and fixed in the 10% formalin solution (prepared in 0.9% normal saline). The fixed adrenal gland samples were given for farther histopathological investigations at Biodiagnostic Centre Parel, Mumbai.

EXPERIMENTAL GROUPS:
Wistar rats (150-200 g) of both sex were divided randomly into fourteen groups , each
containing eight rats.
Group I: Rats received 0.1% Na CMC in Group IX-X I:Rats were treated with
vehicle (Equi volume) (Vehicle control group) MKHA at doses of 50,100 and 200 mg/kg
resp. p.o. and subjected to restraint stress
Group II: Rats received 0.1% Na CMC in Group XH-XIV: Rats were treated with
vehicle and subjected to restraint stress (stress MKM at doses of 50,100 and 200 mg/kg
control group resp.p.o. and subjected to restraint stress
Group in : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to restraint stress (Standard Group)
Group IV : Rats were treated with standard Diazepam (1 mg/kg p.o.) and subjected to restraint stress (Standard Group)
Group V : Rats were treated with standard Piracetam (200 mg/kg p.o.) and subjected to restraint stress (Standard Group)
Group VIVIII: Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o and subjected to restraint stress

Results:

Day l Day 2 Day l4
Treatment groups Mean Serum
corticosteron
e levels
(ng/dl)
±SEM Percentage Decrease Mean Serum
corticostero
ne levels
(ng/dl)
±SEM Percentage Decrease Mean Serum corticoster one levels (ng/dl) ±SEM Percentage Decrease
Vehicle control 11.83 ±1.08 10.19 ±1.31 9.70 ±0.56
Stress control 24.76s ±4.14 109.41 27.75s ±6.84 172.39 28.10* ±4.43 189.69
Diazepam
lmg/kg 21.35 ±2.00 13.78 20.65
±4.25 25.59 20.18 ±2.49 28.20
Ashwagandha 100 mg/kg 20.94 ±4.15 15.45 17.71 ±4.94 36.17 13.43** ±2.90 52.22
MKAQ50 mg/kg 25.00 ±3.25 -0.96 22.79 ±2.31 17.88 21.49 ±2.13 23.53
MKAQ
lOOmg/kg 23.76
±2.53 4.04 21.50 ±2.90 22.52 19.86 ±2.20 29.31
MKAQ
200mg/kg 23.06 ±3.49 6.87 22.41
±2.55 19.23 19.90 ±3.03 29.18
MKHA50 mg/kg 23.40
±3.55 5.50 21.59 ±3.37 22.21 20.99 ±2.41 25.31
MKHA100 mg/kg 24.50 ±4.01 1.06 21.55 ±3.77 22.34 17.44* ±2.33 37.94
MKHA200 mg/kg 24.00 ±2.41 3.08 19.05 ±2.67 31.35 16.48* ±2.05 41.37
MKM50
mg/kg 26.88 ±3.32 -8.53 22.20 ±2.75 20.00 20.16
±2.37 28.27
MKM100 mg/kg 24.10 ±1.83 2.68 19.25 ±1.74 30.63 16.51* ±1.39 41.24
MKM200
mg/kg 25.01
±2.55 -1.01 17.11
±2.45 38.33 15.39** ±2.01 45.24
Values are expressed as mean ± SEM at n=6-8
Statistically significant **=p<0.01,*=p<0.05 when compared with stress control group #=p<0.001,$=
p<0.01 when compared with vehicle control group
The numerical results are evaluated by application of One-way ANOVA with post Dunnet's test for
statistical significance.
TABLE : EFFECT OF EXTRACTS OF MK ON SERUM CORTICOSTERONE
LEVELS IN RS

1. EFFECT OF MK ON SERUM CORTICOSTERONE LEVELS IN RS (Refer Tables: 4.73,4.74 and Figures : 4.65,4.66)
♦ In vehicle control group mean serum CORT levels of 11.83,10 9 and 9.7 ng/ml were observe don 1st ,7th and 14th days of study respectively.
♦ Chronic Restraint Stress produced significant (P<0.0001) increase of 109.41% and 172.39% , 189.69 % in the level of mean serum CORT in stress control group in contrast to vehicle group on 1st, 7th & 14th day of study respectively, thus mean serum corticosterone levels were found to be increased with duration of stress.
♦ Standard AhwagandhalOOmg/kg p.o. produced time dependent decrease in levels of serum CORT on day 1 only where 15.45% (p>0.05)decrease was observed which was increased to 36.17 % (p>0.05) on 7thy day of study and on 14th day of treatment mean serum CORT levels were found to be decreased significantly (p<0.01) by 52.22% as compared to stress control group
♦ Standard Diazepam lmg/kg p.o. produced 13.78% (p>0.05), 25.59 % (p>0.05),and 28.10 %(p>0.05),decrease in mean serum CORT levels on 1SI ,7* and 14th day of study respectively thus indicating that decrease in mean serum CORT levels was increased with duration of treatment up to 7th day but on 14th day no further significant (p>0.05) increase was observed .
♦ MKAQ at dose 100 and 200mg/kg (p.o) produced ceiling effect on 14th day of treatment by producing 29% of inhibition at both the doses as mean serum CORT levels of 21.49ng/ ml and 19.86 ng/ml were observed respectively ,but these inhibitions in stress induced elevated levels of serum CORT as compared with stress control group were not found to be statistically significant (p>0.05) Whereas at dose 50mg/kg (p.o.) on 14th day of study produced 23.53% decrease in mean serum CORT levels which was also not found to be statistically significant (P>0.05) as compared to stress control group.
♦ MKHA at dose 200mg/kg (p.o.) produced 3.08% (p>0.05) ,31.35 % (p>0.05) and significant (P < 0.05) reduction of 41.37 % on 1st ,7th and 14th day of study respectively in mean serum CORT levels as compared to stress control group, at

dose lOOmg/kg (p.o.) 1.06% (p>0.05) 22.34 % (p>0.05) and moderately significant (p<0.05) decrease of 37.94 %was observed on 1st ,7th and 14th day of study respectively as compared to stress control group. At dose 50mg/kg (p.o.) 5.5% (p>0.05),22.21% (p>0.05) and 25.31% (pX).05) decrease in mean serum CORT levels were observed on 1st 7th and 14th day of treatment respectively as compared to stress control group. ♦ MKM at dose 200 mg/kg (p.o.) produced 38.33% decrease in mean serum CORT levels on 7th day which was not found to be statistically significant (p>0.05) whereas 45.24 % decrease in mean serum CORT levels on 14th day of study produced significant (P < 0.01) decrease of 45.24% as compared to stress control group At dose 100 mg/kg (p.o.) 30.63% decrease in mean serum CORT levels was observed on 7th day of study which was not found to be statistically significant (p>0.05) however on 14th day of study moderately significant (p<0.05) decrease of 41.23% was observed as compared to stress control group. Whereas 20% and 28.27 % decrease in mean serum CORT levels observed on 7th and 14th day of study respectively were not found to be statistically significant (p>0.05) as compared to stress control group. MKM did not produce any significant decrease in mean serum CORT levels on 1st day at doses 50 100 and 200mg/kg (p.o) respectively as -8.53% , 2.68 % and -1.01 % of decrease respectively were observed in mean serum CORT levels which were not found to be statistically significant (p>0.05) as compared to stress control group.
Restraint stress is a very commonly used model to assess antistress effect of drugs It is a combination of physical (immobilisation ) and psychological stress. Stress in rats brings about transient activation of the HPA axis, as measured by increased adrenal gland weight with subsequent increase in plasma coiticosterone level and other correlates of adrenal activation which prepares the organism for threatened homeostasis [633]. The important biochemical changes in plasma under stressful conditions, i.e. elevated coiticosterone is necessary to maintain the energy balance which include increased plasma glucose, and decreased triglyceride and cholesterol levels [634]. Under stressful condition adrenal cortex secrets Cortisol in man and corticosterone in rats. Hypersecretion

of Cortisol helps in maintenance of internal homeostasis through the process of gluconeogenesis and lipogenesis. Suppressant effect of MK extract on hyperactivity of adrenals might be responsible for preventing rise in serum corticosterone levels induced by restraint stress. This normalizing effect on plasma corticosterone is one of the possible reasons for their anti stress properties.
References:
l.G.P. Chrousos, Stressors, stress, and neuroendocrine integration of the adaptive response. The 1997 Hans Selye Memorial Lecture, Ann.N.Y. Acad. Sci. 851 (1998)311-335
2Thomas, Samuel, Mathew,Baby, P. Sakaria, Medicinal plants, Kerala Agricultural Unniversity„Aromatic and Medicinal plant research center, 1998, 87-88
3 .Fiebig M Pezzutp J M , Soejarip P P and Kingham AD koeline A future cytotoxic carbazole alkaloid from Murraya Koenigii phytochemishy 1985,24: 3041-3043,
4. Achyut Narayan Kesari, Rajesh Kumar Gupta, Geeta Watal Hypoglycemic effects of Murraya koenigii on normal andalloxan-diabetic rabbits Journal of Ethnopharmacology 97(2005)247-251
5.Dixit V.K. and Sharma A.K., Hypoloipidemic activity of MurrayaKoenigii in rats ,Indian drugs May 2008 ,45 (5): 401-406
6 B. S. Joshi, V. N. Kamat and D. H. Gawad on the structures of girinimbine,mahanimbine, isomahanimbine, koenimbidine and murrayacine Tetrahedron. 1970 Vol. 26: 1475-1482.

CLAIMS:
We claim:
1. A method of isolating and extracting carbazole alkaloids-Mahanimbine from leaves of
Murraya koenigii comprising the steps of:
a) drying and powdering the leaves of Murraya koenigii,
b) extracting the powder of Murraya koenigii obtained in step (a) with methanol.
c) acidifying the above extract with glacial Acetic acid.
d) evoporating the solvent from the above step (c).
e) adding water to the resultant semisolid obtained in the above step (d) and filtering
f) adjusting the pH of the filterate obtained from the above step (e)to acidic .
g) subjecting the above filtrate (f) to liquid extraction with Methyl Isobutane
Ketone (MIBK)
h) separating the two phase obtained in the above step (g) and discarding the aqueous
phase i) collecting and concentrating the Methyl Isobutane ketone (MIBK) phase as an
alkaloid by a method of chromatography.
2. The method of isolating and extracting carbazole alkaloids from leaves of Murraya
koenigii as per claim 1 wherein the method of chromatography used for concentrating in
step (i)comprises of following steps:

a) chromatographing a total 14 gm of MIBK fraction where every time 1 gm of Methyl Isobutane ketone (MIBK) fraction was chromatographed on column (100 cm x 1.3cm) of silica gel (60/120 #).
b) collecting Chloroform elutes (5ml) and following the progress of chromatogram by TLC
c) collecting Fraction 16- 27 (Eluent .chloroform) during each chromatogram and combining
d) distilling off the solvent .
e) crystallization of the residue from hexane to obtain Mahanimbine
f) performing column chromatography repeatedly to obtain sufficient yield of
isolated carbazole alkaloid Mahanimbine.
3.A method of treating stress in mammals including humans by administration of methanolic extract of Murraya koenigii leaves.
4. A method of treating disorders related to elevated plasma levels of corticosterone by administration of effective dose of methanolic extract of Murraya koenigii leaves.