INTRODUCTION:

Essential oils are considered as complex mixture of low molecular weight volatile hydrocarbon and their oxygenated derivatives. They are secondary metabolites extracted from plants by various methods of distillation (steam hydro-distillation etc). They are composed of hydrocarbons and their oxygenated derivatives at different concentrations¹. The characteristic aroma and biological properties of essential oil are due to the presence of major constituents terpenoids and phenylpropanoids². Essential oils derived from plants possess numerous applications in health, food and cosmetics, agriculture etc. they play important role as flavoring agents in beverages food products cosmetics and perfumes^3,4,5. They are stored in oil and resin ducts, glands or trichomes of the plants⁶.

Essential oils are known to exhibits wide range of biological properties including anticancer, antiviral antimicrobial^8,9 anti-mutagenic, antioxidant anti-inflammatory, immunomodulatory, and anti protozoal^10-16. Essential oils have also been used satisfactorily for controlling insects¹⁷. Essential oil from cinnamom clove and rosemary known to possesses antibacterial activity against Streptococcus mutans¹⁸.

Climatic and geographical factors (climatic variations, growth stages, genetic factors and ontogeny) largely affect the composition of essential oil, its yield and its various biological properties. So the information related to chemical polymorphism of a plant can be obtained from its chemical composition in relation to climatic and geographical factors. It can be an important criterion for marketing of essential oil and its commercialization in pharmacy and food technology^19,20,21,22.

Murraya koenigii commonly known as “Curry patta” is aromatic plant of Rutaceae family. The plant is found in tropical regions of Western Africa and all over the world ²³ and is well known for its medicinal importance in treatment of ailments^24,25. The leaves and roots are bitter and have cooling effect, used as antioxidant²⁶anthelmintic, analgesics and to cure piles, thirst, inflammation, itching and also used to cure vomiting and dysentery²⁷. The plant is considered to be important part of Indian kitchen from ancient time due to its characteristics strong aroma and flavor which is due to the presence of sabinene, caryophyllene, cadinol, and cadinene in the essential oil²⁸.

The leaves of Curry patta are immense source of carbohydrates protein carotene, and minerals. Beside this it is known to possess various carbazole alkaloids (girinimbine, murrayafoline-A, mahanimbine and murrayanin), glycosides, isomahanimbine, and furocoumarins^29,30,31,32. Mahanimbine and koenigine obtained from M. koenigii leaves showed radical scavenging activity³³. Carbazole alkaloids mahanine and mahanimbicine isolated from M. koenigii inhibited antibiotic resistant bacteria and suppress the proliferation of cells³⁴. Dichloromethane extracts of Murraya koenigii showed significant antifeedant effect against stored grain pest, Tribolium castaneum³⁵.

MATERIALS AND METHODS:

Plant material:

The freshly collected leaves of Murraya koenigii with significant altitudinal variation were collected in the month Oct-Nov 2017 from three different regions of north India. The regions are Dehradun (2,185 ft), Delhi (744 ft), Mandi (HP) (2790 ft).

Isolation of essential oils:

The fresh leaves of Murraya koenigii collected from different region viz. Dehradun (280g), Mandi (HP) (360g) and Delhi (100g) were crushed subjected to hydro distillation for 6 h for isolation of volatile oil. The collected essential oil was dried by using anhydrous sodium sulphate and was stored in the air tight glass tubes at 5°C for analysis by GC-MS and for testing antifeedant and antimicrobial activities.

GC-MS Analysis:

Analysis by Gas Chromatography was done by using HP 6890 gas chromatograph with HPS fused silica column (30mm X. 32mm X.25μm film thickness) and FID detector. The carrier gas used during analysis was nitrogen.

The oven temperature of column was maintained between 55°C to 225°C with an increase in rate of 3^oC/m. the injector and detector temperature was maintained at 205°C and 220°C respectively Analysis were carried out on a Perkin Elmer Claurus 500 gas chromatograph coupled with Perkin Elmer mass spectrometer. The carrier gas nitrogen was flowed at a rate of 1ml/min. the constituents of oil were identified by using GC retention indices by comparing their mass spectra by reported data in literature³⁶ and by comparing with NIST and WILEY libraries.

Antifeedant activity:

Testing material:

Larvae of Spodoptera litura L. collected from field and were cultivated in the laboratory at 24+2^oC. For antifeedant assay third instar larvae were used from laboratory culture in the present study.

Assay of Antifeedant Activity: The dual choice leaf disc method:

The isolated essential oil from Murraya koenigii was tested against third instar larvae of Spodoptera litura L. (Lepidoptera) and dual choice leaf disc method was used ^37,38,39,40. The leaves of Ricinius communis were washed and taken in the circular discs having area 160cm². The medium vain of the leaves act as a marker for two equal halves. 2.5μg/cm²concentrations of Essential oils isolated from different locations were sprayed on half of circular leaf disc.

Azadirachtin A was used as control^41,42. Leaves were dried and placed in 15cm a Petri dish. Five freshly mounted larvae were positioned at the center of the leaf and examine to feed it for 48 h, for every sample three replicate were maintained. After 48 h the estimation was completed by utilizing ΔT region estimation meter for the unfed region in the treated and for control parts. Percentage feeding index (PFI) was determined as:

% Area fed in treated

PFI= ----------------------------------------------- X 100

% Area fed in treated + % fed in control

Antimicrobial assay:

Microbial strains:

Murraya koenigii essential oil were tested for antimicrobial activity against two Gram-positive bacteria Bacillus cereus S. aureus and two Gram negative bacteria E coli and Proteus mirabilis

Antimicrobial assay: Disc Diffusion method:

Antibacterial activities of the essential oils of Murraya koenigii were tested against Bacillus cereus, S. aureus, E. coli and Proteus mirabilis, by disc diffusion method^43,
44,45,46. The 8mm circular discs made up of Whatman No.1 were used for the assay. 20μL of essential oil was diluted with two volumes of 5% dimethyl sulfoxide (DMSO) and on the circular discs. With the help of sterile forceps placed on plate surface for inoculation Discs sprayed with solvent only act as negative control. Ciprofloxacin (5μg/disc) was used as positive reference standard to test the sensitivity of strain. After incubation of 36h zone of inhibition in the discs were measured. Each experiment was repeated 3 times. By taking mean zone of inhibition was calculated.

RESULT AND DISCUSSION:

The oils were obtained by hydro distillation of Murraya koenigii fresh leaves from three regions of different altitude for 4.5 hours to obtain colorless pleasant smelling oils which was placed in anhydrous sodium sulphate to remove water. The highest oil yield was achieved in case of Mandi sample (0.32%) followed by Dehradun sample (0.25%) and Delhi 0.16%. The gas chromatogram of essential oil showed the presence of 150 components whose percentage varied from almost 35% to 0.001% of TIC (total ion current). Constituents from volatile oil were recognized on the basis of standard samples, retention indices and by correlation of the mass spectral patterns with the data depicted in commercial libraries like NIST and Wiley.

The chemical composition of Murraya koenigii varied from region to region. A comparative analysis of the composition of oil from three distinct areas demonstrated almost similar overall compositions showed differences in percentage of components.

The major identified components from the essential oil from Dehradun were sabinene (32.695%) α-pinene (26.077%), caryophyllene (8.257%), β-pinene (6.307%), D-limonene (3.387%), γ-terpinene (2.448%), aromadendrene (1.762%), d-cadanine (1.648%) and β-myrcene (1.255%). Major components of essential oils from Mandi (HP) region are α-pinene (34.05%), sabinene (27.377%), β-pinene (9.159%), caryophyllene (6.480%), D-limonene (5.128%), γ-terpinene (2.779%), aromadendrene (1.327%) β-myrcene (1.524%), and α-guaiene (1.412%). Major components of essential oils from Delhi are sabinene (25.236%) α- pinene (23.214%), caryophyllene (5.236%), β-pinene (5.215%), D-limonene (3.415%), γ-terpinene (2.122%), aromadendrene (1.023%), and β-myrcene(1.025%). α-pinene, is present in maximum concentration in essential oil from HP 34.058% while sabinene was in essential oils of Dehradun and Delhi samples. The essential oil components are listed in table1.

Table 1: Percentage composition of the essential oils of leaves of Murraya koenigii from three different regions

S.No	RT	Characterized as	Dehradun % area	Mandi (HP) % area	Delhi % area
1	10.439	α-thujene	0.597	0.642	0.432
2	10.829	α-pinene	26.077	34.058	23.210
3	11.349	Camphene	0.391	0.658	0.241
4	12.349	Sabinene	32.695	27.373	25.23
5	12.489	β-pinene	6.730	9.159	5.840
6	12.850	β-myrcene	1.255	1.524	0.988
7	14.000	4-carene	0.896	0.975	0.725
8	14.540	D-limonene	3.387	5.128	3.415
9	15.270	para-cymen-8-ol	0.555	0.642	0.512
10	15.810	γ-terpinene	2.448	2.799	2.120
11	17.150	trans- piperitol	0.584	0.592	0.495
12	17.660	terpinyl acetate	0.249	0.314	0.230
13	26.112	bornyl acetate	0.610	0.817	0.523
14	28.763	α- copaene	0.059	0.046	0.023
15	30.593	β-elemene	0.673	0.402	0.320
16	31.307	α-gurjunene	0.497	0.497	0.452
17	31.903	Caryophyllene	8.257	6.480	5.236
18	33.264	aromadendrene	1.762	1.327	1.023
19	34.364	d- cadanine	1.648	0.600	0.599
20	34.994	α-guaiene	0.551	1.412	0.512

The data obtained from experiments indicated that the essential oil possess significant antibacterial activity against all the clinically isolated bacterial strains. Gram positive bacteria were more sensitive to the oil than the Gram negative bacteria S aureus was the most sensitive strain with the inhibition zone of 14.1 to 14.5mm by all the three samples while the least inhibition zone was shown by Proteus mirabilis 7.8-8.1mm.

CONCLUSION:

There are several factors which can change the chemical composition of a plant. Altitude is one of the important environmental factors which can make a significant impression on the growth of plant. An increase or decrease in altitude can change the temperature, relative humidity and wind speed, availability of water and sunlight which can change the overall chemical composition of plant⁴⁷.

Essential oil was extracted by hydro distillation of leaves of the plant Murraya koenigii collected from three locations in North India, having significant difference in altitude. The highest yield of essential oil (0.32%) was obtained for samples collected from Mandi Himanchal Pradesh (location with highest altitude). This was followed by sample from Dehradun (0.25%). Least yield of essential oil 0.16% was found in Delhi sample (location having lowest altitude). sabinene (32.7%) was the major constituent found in the EO from Dehradun and Delhi region while α- pinene (34.0%), was found in maximum concentration in EO from Mandi, HP. Maximum Antifeedant activity of essential oil was shown by the oil isolated from Mandi (HP) region of highest altitude whereas essential oil isolated from Delhi sample showed minimum antifeedent activity. For antimicrobial activity, Gram positive bacteria were found to be more sensitive to the oil than Gram negative bacteria. S. aureus was most sensitive bacterial strain and showed maximum antimicrobial activity in oil of highest altitude (HP region) whereas its least inhibition zone was found in Delhi sample.

Thus the comparative study of composition of oils from three different regions showed almost similar overall compositions but significant differences in relative yield of components.

Despite the significant variation in the yield of different components of essential oil the high altitude population showed no significant changes in Antifeedant and antimicrobial activity carried out on Essential oils. Thus, altitude seems to be an important factor influencing the yield and the chemical profile of Murraya koenigii.

ACKNOWLEDGEMENT:

The authors are thankful to Centre for Aromatic Plants (CAP), Dehradun for GC-MS Analysis of samples. The authors are also thankful to Dr. Vinod Kumar, Coordinator, Microbiology Lab, Uttaranchal University, Dehradun for antimicrobial activity.

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Received on 19.02.2020 Modified on 08.04.2020

Research J. Pharm. and Tech. 2020; 13(12):5953-5957.

DOI: 10.5958/0974-360X.2020.01039.2

S. No	Particular	%Feeding index (PFI) (2.5μg/cm²)
1	Essential oil (Dehradun)	45.24±3.02
2	Essential oil (Mandi, HP)	40.01±4.22
3	Essential oil (Delhi)	48.53±2.24
4	Azadirachtin A	18.89±4.39

S. No	Particular	(Dehradun sample) Zone of Inhibition (mm)	(HP sample) Zone of Inhibition (mm)	(Delhi sample) Zone of Inhibition (mm)	Ciprofloxacin Zone of Inhibition (mm)
1	Bacillus cereus	11.2±0.5	11.8±0.5	10.7±0.5	23 .5±0.5
2	Staphylococcus aureus	14.2±0.5	14.5±0.5	14.2±0.5	27.3±0.5
3	Escherichia coli	10.3±0.5	10.7±0.5	10.0±0.5	24.3±0.5
4	Proteus mirabilis	7.9 ±0.5	8.1±0.5	7.8 ±0.5	21.8±0.5