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1   2   3   4

‘terpinen-4-ol’

 

and



 

‘1,8-cineole’

 

chemotype



 

(Brophy,


 

1999;


 

Butcher


et

 

al.,



 

1992;


 

Park


 

et

 



al.,

 

2011;



 

Southwell

 

et

 



al.,

 

1992



).

 

However,



 

the


essential

 

oil



 

of

 



M.

 

linariifolia



 

from


 

Brazil


 

was


 

shown


 

to

 



be

 

charac-



terized

 

by



 

methyleugenol

 

(86.8%)


 

and


 

(E)-methylisoeugenol

 

(1.4%)


(

Silva


 

et

 



al.,

 

2010



).

 

A



 

review


 

of

 



the

 

literature



 

revealed


 

that


 

the


medicinal

 

and



 

aromatic


 

potentials

 

of

 



M.

 

linarrifolia



 

growing


 

in

 



India

have


 

not


 

been


 

explored


 

till


 

date.


 

Therefore,

 

in

 



the

 

present



 

research,

chemical

 

compositions



 

and


 

antibacterial

 

potential



 

of

 



essential

 

oil



derived

 

from



 

fresh


 

leaves


 

of

 



M.

 

linarrifolia



 

grown


 

in

 



the

 

foothills



 

of

northern



 

India


 

has


 

been


 

investigated.

2.

 

Materials



 

and


 

methods


2.1.

 

Plant



 

materials

 

and


 

isolation

 

of

 



essential

 

oil



Fresh

 

leaves



 

of

 



M.

 

linarrifolia



 

were


 

collected

 

from


 

plant


 

grown


 

at

Pantnagar



 

(latitude

 

29.02


N,

 



longitude

 

79.31



E

 



and

 

an



 

altitude


 

of

237



 

m)

 



in

 

Udham



 

Sing


 

Nagar


 

of

 



Uttarakhand,

 

India.



 

The


 

plant


 

mate-


rial

 

was



 

authenticated

 

at

 



Taxonomy

 

Department



 

of

 



CSIR-Central

Institute

 

of

 



Medicinal

 

and



 

Aromatic


 

Plants,


 

Research


 

Center,


 

Pant-


nagar,

 

by



 

one


 

of

 



the

 

authors



 

(Amit


 

Chauhan).

 

Voucher


 

specimen


and

 

herbarium



 

record


 

of

 



the

 

plant



 

have


 

been


 

retained


 

at

 



CIMAP

Research


 

Centre,


 

Pantnagar.

 

Fresh


 

leaves


 

of

 



the

 

plant



 

were


 

sub-


jected

 

to



 

hydrodistillation

 

in

 



a

 

Clevenger’s



 

type


 

apparatus

 

for


 

3

 



h,

for


 

isolation

 

of

 



essential

 

oil.



 

Essential

 

oil


 

was


 

measured


 

directly


 

in

the



 

extraction

 

burette,


 

and


 

contents


 

(%)


 

were


 

calculated

 

as

 



volume

(mL)


 

of

 



essential

 

oil



 

per


 

100


 

g

 



of

 

fresh



 

weight


 

of

 



plant

 

material.



The

 

crude



 

oil


 

was


 

dehydrated

 

over


 

anhydrous

 

Na

2



SO

4

(SD



 

Fine-


Chem

 

limited,



 

Mumbai,


 

India)


 

and


 

kept


 

in

 



a

 

cool



 

and


 

dark


 

place


until

 

further



 

analyses.

2.2.

 

Analysis



 

of

 



essential

 

oils



GC

 

analysis



 

of

 



the

 

essential



 

oil


 

was


 

carried


 

out


 

on

 



a

 

Perkin



 

Elmer


AutoSystem

 

XL



 

gas


 

chromatograph

 

equipped


 

with


 

flame


 

ioniza-


tion

 

detector



 

(FID)


 

and


 

a

 



DB-5

 

capillary



 

column


 

(60


 

m

 



×

 

0.32



 

mm

i.d.,



 

film


 

thickness

 

0.25


 

␮m).


 

The


 

oven


 

column


 

temperature

 

ranged


from

 

70



 

to

 



250

C,



 

programmed

 

at

 



3

C



 

min


−1

,

 



with

 

initial



 

and


 

final


hold

 

time



 

of

 



2.0

 

min,



 

using


 

H

2



as

 

carrier



 

gas


 

at

 



1.0

 

mL



 

min


−1

.

 



Injec-

tor


 

and


 

detector


 

(FID)


 

temperatures

 

were


 

set


 

at

 



250

 

and



 

280


C,

respectively.



 

Injection

 

size


 

was


 

0.02


 

␮L

 



(neat

 

oil),



 

with


 

a

 



split

 

ratio



1:35.

 

GC-MS



 

analysis


 

of

 



the

 

essential



 

oils


 

were


 

performed

 

on

 



a

Clarus


 

680


 

GC

 



interfaced

 

with



 

a

 



Clarus

 

SQ



 

8C

 



mass

 

spectrometer



of

 

Perkin



 

Elmer


 

fitted


 

with


 

Elite-5


 

MS

 



fused-silica

 

capillary



 

column


(30

 

m



 

×

 



0.25

 

mm



 

i.d.,


 

film


 

thickness

 

0.25


 

␮m;


 

Supelco


 

Bellefonte,

PA,

 

USA).



 

The


 

oven


 

temperature

 

program


 

was


 

from


 

60

 



to

 

240



C,

 



at

3



C

 

min



−1

,

 



and

 

programmed



 

to

 



270

C



 

at

 



5

C



 

min


−1

;

 



injector

 

tem-



perature

 

was



 

250


C;

 



transfer

 

line



 

and


 

source


 

temperatures

 

were


220

C;



 

injection

 

size


 

0.03


 

␮L

 



(neat

 

oil);



 

split


 

ratio


 

1:50;


 

using


 

He

as



 

carrier


 

gas


 

at

 



1.0

 

mL



 

min


−1

;

 



ionization

 

energy



 

70

 



eV;

 

mass



 

scan


range

 

40–450



 

amu.


2.3.

 

Identification



 

of

 



essential

 

oil



 

constituents

Identification

 

of



 

essential

 

oil


 

constituents

 

was


 

accomplished

on

 

the



 

basis


 

of

 



retention

 

index



 

(RI,


 

determined

 

with


 

reference

 

to

homologous



 

series


 

of

 



n-alkanes,

 

C



8

–C

24;



Supelco

 

Analytical,



 

Belle-


fonte

 

PA,



 

USA,


 

under


 

same


 

temperature-programmed

 

conditions),



co-injection

 

with



 

standards

 

(Aldrich


 

and


 

Fluka),


 

reference

 

mass


spectral

 

library



 

search


 

(NIST/EPA/NIH

 

version


 

2.1


 

and


 

Wiley


 

reg-


istry

 

of



 

mass


 

spectral


 

data


 

7

th



edition),

 

and



 

by

 



comparison

 

of



 

mass


spectra

 

data



 

with


 

literature

 

(

Adams,



 

2007


).

 

The



 

relative


 

content


 

of

individual



 

components

 

of

 



the

 

oil



 

is

 



expressed

 

as



 

percent


 

peak


 

area


relative

 

to



 

total


 

peak


 

area


 

of

 



the

 

GC/FID



 

chromatogram

 

automated



electronic

 

integration



 

without


 

response


 

factor


 

correction.

2.4.

 

Antibacterial



 

assay


The

 

antibacterial



 

activity


 

of

 



the

 

essential



 

oil


 

was


 

determined

by

 

filter



 

paper


 

disc


 

diffusion

 

assay


 

(

Bauer



 

et

 



al.,

 

1966



).

 

Inoculums



of

 

the



 

test


 

bacteria


 

[Gram-positive:

 

Staphylococcus



 

aureus


 

(MTCC


96),

 

S.



 

aureus


 

(MTCC


 

2940),


 

Streptococcus

 

mutans


 

(MTCC


 

890),


 

S.

epidermidis



 

(MTCC


 

435),


 

Bacillus


 

subtilis


 

(MTCC


 

121),


 

and


 

Gram-


negative:

 

Klebsiella



 

pneumoniae

 

(MTCC


 

109),


 

Escherichia

 

coli


 

(MTCC


723),

 

Pseudomonas



 

aeruginosa

 

(MTCC


 

741),


 

Salmonella

 

typhimurium



(MTCC

 

98)



 

was


 

prepared


 

equivalent

 

to

 



McFarland

 

Standard



 

0.5.


Uniform

 

bacterial



 

lawns


 

were


 

made


 

using


 

100


 

␮L

 



inoculums

 

on



 

a

nutrient



 

agar


 

plate.


 

Filter


 

paper


 

(Whatman)

 

discs


 

(5.0


 

mm)


 

soaked


with

 

test



 

essential

 

oil


 

were


 

placed


 

over


 

seeded


 

plates.


 

The


 

plates


were

 

incubated



 

at

 



37

C



 

for


 

24

 



h.

 

Activity



 

was


 

measured


 

in

 



terms

 

of



zone

 

of



 

inhibition

 

(mm).


 

The


 

net


 

zone


 

of

 



inhibition

 

was



 

determined

by

 

subtracting



 

the


 

disc


 

diameter


 

(5.0


 

mm)


 

from


 

the


 

total


 

zone


 

of

inhibition



 

shown


 

by

 



the

 

test



 

disc


 

in

 



terms

 

of



 

clear


 

zone


 

around


 

the


disc.

 

The



 

tests


 

were


 

performed

 

in

 



triplicate.

 

The



 

bacterial

 

strains


were

 

obtained



 

from


 

the


 

Microbial

 

Type


 

Culture


 

Collection

 

Centre


(MTCC),

 

Institute



 

of

 



Microbial

 

Technology



 

(IMT),


 

Chandigarh,

 

India.


2.5.

 

Minimum



 

inhibitory

 

concentration



 

(MIC)


Antibacterial

 

activity



 

of

 



essential

 

oils



 

was


 

determined

 

by

 



Micro

dilution


 

broth


 

assay


 

using


 

96

 



‘U’

 

bottom



 

micro-titer

 

plates


 

as

per



 

CLSI


 

guidelines

 

(

CLSI,



 

2012


).

 

Samples



 

were


 

serially


 

diluted


twofolds

 

(in



 

the


 

range


 

of

 



1000–1.95

 

␮g



 

mL

−1



)

 

in



 

Mueller


 

Hinton


Broth

 

(MHB).



 

The


 

broth


 

was


 

inoculated

 

with


 

10.0


 

␮L

 



of

 

diluted



24

 

h



 

grown


 

culture


 

of

 



test

 

organisms



 

with


 

a

 



titer

 

equivalent



 

to

 



0.5

McFarland

 

standards.



 

The


 

inoculated

 

plates


 

were


 

then


 

incubated

at

 

37



C

 



for

 

16–24



 

h

 



and

 

the



 

growth


 

was


 

recorded


 

spectropho-

tometrically

 

at



 

600


 

nm

 



using

 

Spectramax



 

190-microplate

 

reader


(Molecular

 

Devices,



 

CA,


 

and


 

USA).


 

The


 

MIC


 

value


 

was


 

determined

from

 

the



 

turbidimetric

 

data


 

as

 



the

 

lowest



 

concentration

 

showing


growth

 

inhibition



 

equal


 

to

 



or

 

greater



 

than


 

80%


 

as

 



compared

 

to



 

con-


trol.

 

Experimental



 

observations

 

were


 

performed

 

in

 



triplicate

 

to



 

rule


out

 

any



 

error


 

during


 

the



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