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Variation in essential oil composition of Ocimumamericanum L. from north-western Himalayan regionS. Singh , G. Tewari a , C. Pande a & C. Singh aa Department of Chemistry , Kumaun University , Nainital , 263 002 , Uttarakhand , IndiaPublished online: 20 Mar 2013.

To cite this article: S. Singh , G. Tewari , C. Pande & C. Singh (2013) Variation in essential oil composition ofOcimum americanum L. from north-western Himalayan region, Journal of Essential Oil Research, 25:4, 278-290, DOI:10.1080/10412905.2013.775079

To link to this article: http://dx.doi.org/10.1080/10412905.2013.775079

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Variation in essential oil composition of Ocimum americanum L. from north-westernHimalayan region

S. Singh, G. Tewari*, C. Pande and C. Singh

Department of Chemistry, Kumaun University, Nainital 263 002, Uttarakhand, India

(Received 8 April 2012; final form 21 January 2013)

The essential oils of ten wild growing Ocimum americanum L. of the family Lamiaceae collected from differentlocations in Uttarakhand, India, were analyzed by capillary gas chromatography (GC) and GC/mass spectrometry(GC/MS). On the basis of chemical composition and cluster analysis, O. americanum was classified into six groups.Group I was significantly rich in methyl chavicol, 1,8-cineole, (E)-γ-bisabolene, β-bisabolene and eugenol, whilegroup II has (E)-γ-bisabolene, aliphatic hydrocarbons, eugenol, β-bisabolene and methyl chavicol as major constitu-ents. Eugenol and (E)-caryophyllene were found in abundant quantity in group III and linalool with methyl chavicolin group IV. Group V contained aliphatic hydrocarbons, eugenol, camphor and 1,8-cineole in high content while cam-phor and aliphatic hydrocarbons were found in group VI. Linalool was positively correlated with methyl chavicol,while eugenol with (E)-α-bisabolene and β-bisabolene with (E)-γ-bisabolene. The essential oil composition of O.amercanum was found to be affected by variation in soil properties and microclimatic conditions.

Keywords: Ocimum americanum L; groups; eugenol; methyl chavicol; camphor; linalool; δ-cadinene; bisabolenes

1. Introduction

Ocimum species (basil) is an annual, aromatic,branched herb, 30–150 cm high. The plant has beenused for medicinal purposes as a digestive stimulantand for treatment of insomnia and constipation. Theoils of basil have been used principally in the food andcosmetic industries (1, 2). Some of the volatile com-pounds of Ocimum have been suggested as allelopathicagents (3). Essential oil of O. americanum has antibac-terial activity against Staphylococcus aureus, Strepto-mycespyo genes, Escherichia coli, Streptococcusmutans, Candida albicans and Salmonella typhos (4).The oil of O. americanum also has antifungal activityagainst a great number of fungi including some humanpathogens (5). On the basis of the available data, it hasbeen found that Ocimum species has various chemo-types. Essential oils compositions of twelve varieties ofbasil grown in Colombia were found with the existenceof three chemotypes (6). Ocimum basilicum alsoshowed chemotaxonomical variations (7, 8) Terpinene-4-ol chemotype has been reported from O. americanumalong with other chemotypes (9–11). The genus Oci-mum has a large number of subspecies and varietiesdue to abundant cross-pollination. To the best of ourknowledge, no work has been done on the chemicalvariability of Ocimum americanum growing wild inUttarakhand. Therefore, the aim of present study wasto classify this species on the basis of chemicalmarkers.

2. Materials and methods

2.1. Plant collection and identification

Fresh plant materials of O. americanum L. at floweringstage along with its soil samples (0–20 cm) were col-lected in August to November 2008 from ten locations,namely Rudrapur (29°30′N, 79°28′E), Nainital (29°23′N, 79°30′E), Banbasa (28°59′N, 80°04′E), Rushi vil-lage (29°23′N, 79°30′E), Champawat (29°36′N, 79°30′E), Almora (29°37′N, 79°40′E), Kilbury (29°23′N, 79°30′E), Dhoulchina (29°37′N, 79°40′E), Mussoorie (30°27′N, 78°06′E) and Dharchula (29°51′00′′N: 80°31′60′′E) in Kumaun Himalaya (Uttarakhand, India). Ocimumamericanum plants were in full blooming stage. Thebotanical identification of the specimen was done at theBotany Department, Kumaon University, Nainital, andsubmitted to the Botanical Survey of India, Dehradun(Voucher no. 33483).

2.2. Physicochemical properties of soil properties

The soil pH and EC was determined in 1:2 soil/watersuspension (12), the soil organic carbon content wasdetermined by the Walkley and Black method and thetexture was determined by the hydrometer method (13).The cation exchange capacity was measured by theNH4

+ saturation method given by Chapman (14). Thetotal metal content of the soils was determined in aquaregia digests (15). The water holding capacity of thesoils was determined by using Hilgard apparatus (16).

*Corresponding author. Email: geeta_k@rediffmail.com

The Journal of Essential Oil Research, 2013Vol. 25, No. 4, 278–290, http://dx.doi.org/10.1080/10412905.2013.775079

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Table

1.Phy

sicochem

ical

prop

ertiesof

soils.

General

soil

prop

erties

Banbasa

Rushi

Champawat

Nainital

Dho

ulchina

Rud

rapu

rDharchu

laAlm

ora

Kilb

ury

Mussoorie

Mechanical

analysis

Sand(%

)76

.0±0

.00

82.0±1

.00

78.0±0

.00

66.0±0

.00

76.0±0

.00

66.0±1

.00

73.0±1

.00

84.0±0

.00

68.0±0

.00

46.0±0

.00

Silt

(%)

18±1

.00

16±0

.00

12±1

.00

20±0

.00

22±0

.00

22±0

.00

26±0

.014

±0.00

26±0

.00

40.0±1

.00

Clay(%

)6.0±

0.00

2.0±

0.00

8.0±

0.00

16.0±0

.00

2.0±

0.00

16.0±0

.00

2.0±

0.00

2.0±

0.00

6.0±

0.00

14.0±0

.00

Texture

Loamysand

Loamysand

Loamysand

Sandy

loam

Sandy

loam

Sandy

loam

Sandy

loam

Loamysand

Sandy

loam

Other

soil

prop

-erties

pH(1:2)

7.44

±0.040

6.00

±0.130

5.61

±0.050

7.09

±0.070

8.2±

0.06

05.83

±0.060

7.17

±0.030

7.32

±0.110

7.41

±0.040

7.65

±.0.33

0EC

0.34

0±0.00

40.01

6±0.00

40.07

4±0.00

90.18

9±0.00

60.02

5±0.00

40.111±

0.00

10.19

5±0.02

10.05

9±0.00

60.32

3±0.00

50.14

2±0.00

4O.C.%

3.26

±0.005

1.54

5±0.00

63.22

8±0.00

63.83

1±0.00

70.66

8±0.011

1.70

6±0.09

83.93

1±0.05

21.20

2±0.00

63.00

4±0.01

54.52

1±0.03

9CEC

42.40±

0.04

044

.51±

0.04

025

.76±

0.09

029

.38±

0.04

014

.81±

0.07

010

.26±

0.05

042

.40±

0.50

019

.72±

0.05

028

.53±

0.05

025

.89±

0,06

0WHC

46.08±

0.07

039

.65±

0.05

039

.57±

0.06

037

.92±

0.05

036

.10±

0.06

053

.28±

0.07

040

.13±

0.06

049

.69±

0.07

058

.51±

0.05

020

.28±

0.05

0Total content

(mg/kg

)Zn

91.677

±0.012

49.466

±0.026

40.5±0

.016

55.307

±0.223

27.600

±0.035

35.05±

0.02

390

.358

±0.005

73.021

±0.094

54.281

±0.012

33.920

±0.021

Fe

559.24

8±0.01

757

2.68

7±0.00

953

6.47

7±0.01

256

0.115±

0.01

049

3.35

0±0.28

055

9.68

2±0.01

355

0.57

8±0.05

155

6.64

7±0.02

552

2.06

4±0.01

552

0.00

0±0.16

0Cu

15.550

±0.012

12.900

±0.063

15.300

±0.053

16.250

±0.013

10.500

±0.031

24.630

±0.014

11.125

±0.037

28.350

±0.079

19.825

±0.029

21.080

±0.110

Mn

348.02

3±0.112

323.89

4±4.00

526

4.39

6±0.02

716

5.90

9±0.47

384

.775

±0.086

160.07

3±0.07

817

1.19

3±0.01

035

2.77

8±0.01

322

0.89

7±0.99

998

.999

±0.301

Available

content

(mg/kg

)Zn

12.262

±0.018

1.32

4±0.00

27.36

8±0.00

62.71

0±0.01

03.95

4±0.02

26.38

4±0.01

08.93

0±0.15

69.45

2±0.02

90.98

4±0.02

01.46

6±0.01

0Fe

57.980

±0.005

35.380

±0.015

28.770

±0.490

22.646

±0.005

16.016

±0.005

36.676

±0.003

95.954

±0.008

29.940

±0.011

91.701

±0.013

35.844

±0.045

Cu

7.32

6±0.01

50.52

6±0.02

31.83

0±0.01

40.15

0±0.02

01.14

8±0.10

21.119±

0.00

80.35

9±0.00

22.25

6±0.02

41.82

0±0.09

00.32

4±0.01

4Mn

17.396

±0.167

6.68

0±0.27

315

.430

±1.260

7.07

6±0.57

72.06

0±0.13

814

.278

±0.471

3.16

0±0.53

014

.560

±0.090

17.108

±1.807

15.169

±0.157

Macronu

-trients(%

)N(av)

0.00

65±0

.00

0.01

45±0

.00

0.00

83±0

.001

0.00

56±0

.002

0.00

47±0

.001

0.01

07±0

.001

0.00

42±0

.00

0.01

03±0

.001

0.00

87±0

.00

224.00

±0.680

N(tot)

0.34

8±0.08

40.27

6±0.04

90.18

0±0.01

50.20

0±0.06

50.16

3±0.00

50.30

0±0.00

70.03

0±0.01

50.23

5±0.05

70.03

6±0.00

20.02

3±0.01

3P

0.00

24±0

.001

0.00

9±0.00

30.00

14±0

.00

0.00

26±0

.001

0.00

25±0

.00

0.00

3±0.00

10.00

3±0.00

20.00

19±0

.00

0.00

28±0

.001

0.00

3±0.00

1K

0.00

90±0

.00

0.01

90±0

.00

0.01

32±0

.00

0.01

63±0

.002

0.00

90±0

.001

0.02

18±0

.008

0.02

44.±0.00

20.01

00±0

.005

0.00

57±0

.002

0.00

60±0

.002

Notes:EC,electrical

conductiv

ity(dS/cm);O.C.%

,organiccarbon

;CEC,catio

nexchange

capacity

(cmol/kg);WHC,water

holdingcapacity;(av),available;

(tot),total.

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Total nitrogen was determined by the Kjeldhal method(17). Available phosphorus in the soil was determinedby Olsen’s method and potassium estimation was doneby using a flame photometer (16). Soil samples wereextracted for DTPA extractable metals following theprocedure developed by Lindsay and Norvell (18). Theproperties of the soil samples taken for this study aregiven in Table 1, and climatic and some other proper-ties are given in Table 2.

2.3. Preparation of plant samples for micronutrientanalysis

The collected plant material were washed sequentiallywith tap water, 0.1 N HCl solution and finally with dis-tilled water. Dry plant tissue was finely ground and wetashed using HNO3:H2SO4:HClO4 (10:1:4 v/v) (19).

2.4. Heavy metal analysis in soil and plant samples

Concentrations of Zn, Cu, Mn and Fe in prepared plantand soil samples were analyzed by atomic absorptionspectrophotometer (GBC-902 and Avanta sigmaModels).

2.5. Isolation of essential oil

One kilogram of fresh aerial plant material was steamdistilled for 3 hours using a copper still fitted withspiral glass condensers and extracted with 80 mLn-hexane and 40 mL dichloromethane. The organicphase was dried over anhydrous sodium sulfate and thesolvent removed by distillation using a thin film rotaryvacuum evaporator at 25–30°C. The oil yield was0.36–0.98% (v/w).

2.6. Analysis of the essential oil

The oil was analyzed by using a Perkin Elmer Autosys-tem XL gas chromatograph (GC). The column tempera-ture was programmed from 70° to 290° at 3°C/minuteusing hydrogen as the carrier gas at 10 psi columnhead pressure. The injector temperature was 220°C anddetector (flame ionization, FID) temperature was290°C. The GC/mass spectrometer (GC/MS) used wasan Autosystem XL GC (Equity-5, 60 m � 0.32 mm �0.25 μm i.d., FID) coupled with a Perkin Elmer Turbo-mass fitted with a fused silica capillary column(Equity-5; 60 m � 0.32 mm � 0.25 μm film thick-ness). The column temperature ranged from 70° to300°C at 3°C/minute using helium as the carrier gas.The injector temperature was 220°C, and the injectionvolume 0.05 μL in n-hexane, with a split ratio of 1:50.MS were taken at 70 eV with a mass range of 40–450amu (oil from Nainital, Banbasa, Rushi, Champawat,Almora, Kilbury, Dhoulchina, Mussoorie andDharchula). Oil from Rudrapur was analyzed by Nucon5765 gas chromatograph (Rtx-5 column, 30 m � 0.32mm, FID), split ratio 1:48, N2 flow of 4 kg/cm2 andT

able

2.Geographicandoilprop

erties.

Microclim

atic

cond

ition

sandotherprop

erties

Banbasa

Rushi

Champawat

Nainital

Dho

ulchina

Rud

rapu

rDharchu

laAlm

ora

Kilb

ury

Mussoorie

Altitude

(m)

375

1620

1650

1950

1800

243

2283

1550

2220

1600

Tem

perature

(°C)

3024

2520

2437

2130

1922

Plant

height

21.0±0

.200

25.0±0

.600

23.5±0

.400

27.5±0

.400

27.5±0

.300

16.0±0

.200

29.5±0

.700

12.5±0

.700

45.3±0

.500

22.5±0

.400

Sun

/shady

side

Sun

nyShady

Sun

nySun

nySun

nySun

nySun

nyShady

Shady

Sun

nyMon

thof

collectionin

2008

September

Aug

ust

September

September

Aug

ust

Aug

ust

Aug

ust

September

September

September

Oilcolor

Darkyello

wYellow

Yellow

Yellow

Light

yello

wDarkyello

wDarkyello

wLight

yello

wYellow

Yellow

Oil%

0.36

0.48

0.54

0.60

0.84

0.42

0.98

0.36

0.60

0.38

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Table

3.Chemical

compo

sitio

nof

Ocimum

american

umcollected

from

differentsites.

S.

No.

Com

poun

dsa

RIb

RIc

Typ

eI

Type

IITyp

eIII

Type

IVTyp

eV

Typ

eVI

Rud

rapu

r,24

3m

Nainital,

1950

mBanbasa,

375m

Rushi,

1620

mChampawat,

1650

mAlm

ora,

1550

mKilb

ury,

2220

mDho

ulchina,

1800

mMussoorie,

1600

mDharchu

la,

2283

m

1α-Thu

jene

(MH)

930

924

––

––

––

0.3

––

–2

α-Pinene(M

H)

939

932

––

––

0.6

–0.4

––

t3

Sabinene(M

H)

975

969

––

tt

0.1

t0.2

–0.3

t4

β-Pinene(M

H)

979

974

t0.2

t0.3

tt

––

1.6

t5

3-Octanone(A

C)

983

979

––

––

0.5

t–

––

–6

β-Myrcene

(MH)

990

988

0.3

–t

0.1

0.2

––

–0.2

0.2

7α-Terpinene

(MH)

1017

1014

–t

–t

––

––

0.3

–8

p-Cym

ene(M

H)

1024

1020

0.2

––

––

0.3

–0.2

–t

9Lim

onene(M

H)

1029

1024

t–

0.2

0.2

0.1

0.2

1.1

–t

0.1

101,8-Cineole

(OM)

1031

1026

t4.5

12.4

10.3

16.8

11.5

28.4

1.2

0.7

t11

(Z)-β-Ocimene(M

H)

1037

1032

t–

––

4.9

–1.0

––

0.6

12(E)-β-Ocimene(M

H)

1050

1044

t1.6

1.2

3.8

–0.2

––

0.4

–13

γ-Terpinene(M

H)

1059

1054

––

––

t–

–0.3

0.8

–14

Linaloo

l(O

M)

1096

1095

tt

––

t2.2

3.0

2.9

0.4

14.2

15(E)-Sabinenehy

drate

(MH)

1099

1098

––

––

0.4

2.8

1.8

––

–

16Cam

phor

(OM)

1146

1141

––

––

–49

.442

.00.5

23.0

–17

δ-Terpineol

(OM)

1166

1162

t–

––

0.2

1.9

0.9

0.4

–18

Borneol

(OM)

1169

1165

t–

t0.2

––

––

0.4

–19

Terpinen-4-ol(O

M)

1177

1174

––

––

0.1

–2.0

–1.0

–20

Iso-Menthol(O

M)

1182

1179

––

––

––

0.5

––

–21

α-Terpineol

(OM)

1188

1186

–0.3

0.4

0.4

0.6

3.8

2.2

1.1

––

22Myrtenol(O

M)

1195

1194

––

––

–3.3

––

0.9

–23

Methy

lchavicol

(PP)

1196

1195

–1.7

10.2

11.7

12.9

–1.3

28.9

–78

.324

Chavicol(PP)

1250

1247

–7.8

0.4

0.8

0.1

–0.5

0.5

0.5

0.3

25Thy

mol

(OM)

1290

1289

––

––

––

–0.3

––

262E

,4Z-D

ecadienal

(OM)

1293

1292

––

––

––

––

1.7

–

27δ-Elemene(SH)

1338

1335

––

–0.2

––

–0.2

––

28Eug

enol

(PP)

1359

1356

45.2

37.8

38.2

31.8

27.6

–8.6

14.7

––

29α-Cop

aene

(SH)

1376

1374

0.8

––

––

––

––

30β-Bou

rbon

ene(SH)

1386

1387

0.5

––

––

––

––

–31

D-Elemene(SH)

1390

1388

––

t–

––

––

0.6

–32

Methyleugenol

(PP)

1400

1403

14.8

––

––

––

––

–33

α-Cedrene

(OM)

1411

1410

t–

––

––

––

––

34Thy

moh

ydroqu

inon

edi

methy

lether(O

M)

1418

-–

––

––

––

0.3

––

35(E)-Caryo

phyllene

(SH)

1419

1417

30.2

1.2

1.0

1.8

2.3

1.0

0.6

1.6

2.4

0.5

36β-Gurjunene

(SH)

1433

1431

t–

––

––

––

–

(Contin

ued)

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Table3.

(Contin

ued)

S.

No.

Com

poun

dsa

RIb

RIc

Typ

eI

Typ

eII

Typ

eIII

Typ

eIV

Typ

eV

Typ

eVI

Rudrapur,

243m

Nainital,

1950

mBanbasa,

375m

Rushi,

1620

mChampawat,

1650

mAlm

ora,

1550

mKilb

ury,

2220

mDho

ulchina,

1800

mMussoorie,

1600

mDharchu

la,

2283

m

37(E)-α-Bergamotene

(SH)

1434

1432

–1.0

1.1

1.5

0.3

––

1.8

–T

38β-(E)-Farnesene

(SH)

1456

1454

–0.8

0.6

1.0

0.7

–0.2

––

0.5

39α-Hum

ulene(SH)

1454

1452

1.6

–1.3

1.6

1.3

–1.1

1.6

–0.3

40γ-Muu

rolene

(SH)

1480

1478

0.6

––

––

–41

Germacrene

D(SH)

1485

1484

0.8

5.4

2.8

4.0

3.6

1.6

0.5

1.4

4.4

0.7

42α-Selinene(SH)

1498

1498

––

––

––

––

–t

43Bicyclogerm

acrene

(SH)

1500

1500

––

––

–0.3

0.8

––

t

44(E),(E)-α-Farnesene

(SH)

1505

1505

t–

0.2

0.2

––

––

–0.1

45β-Bisabolene(SH)

1505

1505

–17

.812

.513

.59.9

–0.7

14.7

––

46(E)-γ-Bisabolene(SH)

1522

1529

t15

.814

.512

.59.8

0.6

–7.1

––

47Spathulenol

(OS)

1578

1577

––

––

–0.3

–0.7

–2.1

48Caryo

phyllene

oxide

(OS)

1583

1582

0.8

t–

0.2

––

–0.8

0.3

–

49Hum

uleneepox

ideII

(OS)

1608

1608

t–

––

––

––

––

5010

-Epi-γ-eud

esmol

(OS)

1623

1622

t–

––

––

––

––

51Epi-α-Cadinol

(OS)

1640

1638

t–

––

––

–0.2

0.2

–52

Cub

enol

(OS)

1646

1645

––

––

t–

––

––

53β-Eud

esmol

(OS)

1649

1650

t–

––

–54

α-Cadinol

(OS)

1654

1652

––

––

––

0.2

0.4

–55

(Z)-α-(E)-Bergamotol

(OS)

1690

1690

––

––

0.4

––

1.0

––

562(E),6(E)-Farnesol

(OS)

1743

1742

–0.3

0.3

0.5

––

––

––

57n-Octadecane(A

C)

1800

1800

––

––

––

––

10.9

–58

n-Non

adecane(A

C)

1900

1900

––

––

––

––

16.0

–59

Eicosane(A

C)

1988

1987

––

––

––

––

17.2

–60

Docosene<1>

(AC)

2182

2189

––

––

4.1

––

––

–61

n-Tetracosane

(AC)

2400

2400

––

––

–1.6

––

––

62Hexacosane(A

C)

2600

2600

––

––

–4.4

0.5

8.4

––

63Octacosane(A

C)

2800

2800

––

––

–5.4

0.6

––

–%

Identified

95.8

96.2

97.3

96.6

97.5

90.8

99.2

91.0

84.6

97.9

Notes:a M

odeof

identifi

catio

n:retentionindex,

coinjectionwith

standards/peak

enrichmentwith

know

noilconstituents.

bRetentio

nindicesdeterm

ined

ontheEquity

-5columnusingan

n-alkane

homol-

ogousseries

(C9–C

24).

c Retentio

nindicesfrom

theliterature(20).Boldtype

indicatesmajor

compo

nents,t,trace(<0.1%

);MH,mon

oterpene

hydrocarbons;OM,ox

ygenated

monoterpenes;SH,sesqui-

terpenes

hydrocarbons;OS,oxygenated

sesquiterpenes;AC,aliphatic

compounds;PP,

phenyl

propanoids.

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Thermo Quest Trace GC 2000 interfaced with FinniganMAT Polaris Q Ion Trap Mass spectrometer fitted witha Rtx-5 (Restek Corp.) fused silica capillary column(30 m � 0.25 mm; 0.25 μm film coating). The columntemperature was programmed from 60° to 210°C at3°C/minute using helium as the carrier gas at1.0 mL/minute. The injector temperature was 210°C,injection size 0.1 μL prepared in n-hexane, split ratio1:40. MS were taken at 70 eV with a mass range of40–450 amu.

2.7. Identification of the components

Identification of constituents were done on the basis ofretention index [RI, determined with reference tohomologous series of n-alkanes (C9–C24, PolyscienceCorp., Niles, IL) under identical experimental condi-tion], co-injection with standards (Sigma) and knownessential oil constituents (standard isolates), MS Librarysearch (NIST: NIH version 2.1 and WILEY: 7thedition), by comparing with the MS literature data (20).The relative amounts of individual components were

calculated based on GC peak area (FID response)without using correction factor.

2.8. Statistical analysis

Experimental data were processed using MicrosoftExcel XP. Correlation coefficients were calculatedamong the major constituents of oil, micro- andmacronutrients, microclimatic conditions and physicalproperties of soil. The significance level of correlationcoefficient was checked on probability levels ofp60.05 and p60.01. Ward’s hierarchical clusteringanalysis of major constituents of essential oils was con-ducted in order to discriminate groups. All statisticalanalyses were performed using SPSS 16.0 and theresulting dendrogram was illustrated.

3. Results and discussion

3.1. Essential oil composition

The essential oil composition of aerial parts of ten sam-ples of O. americanum L., family Lamiaceae, collected

Figure 1. Gas chromatogram of the essential oil of Ocimum americanum L. from Banbasa.

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from different locations in Central Himalayas, India,was analyzed by GC (Figure 1–6) and GC/MS. Ward’shierarchical clustering analysis of major constituents ofessential oils was conducted in order to classify groups.The result of cluster analysis showed six groups on thebasis of difference in their main chemical constituents,allowing them to be characterized into six distinctgroups (Table 3, Figure 7). The oils from Nainital,Banbasa, Rushi and Champawat (group I) showed thepresence of 1,8-cineole (4.5–16.8%), methyl chavicol(1.7–12.9%), eugenol (27.6–38.2%), β-bisabolene(9.9–17.8%) and (E)-γ-bisabolene (9.8–15.8%), whilemethyl chavicol (28.9%), eugenol (14.7%), β-bisabo-lene (14.7%), (E)-γ-bisabolene (7.1%) and aliphatichydrocarbons (8.4%) were the major constituents in theDhoulchina oil (group II). The oil from Rudrapur(group III) was rich in eugenol (45.2%), methyleugenol(14.8%) and (E)-caryophyllene (30.2%) as major con-stituents, while the oil collected from Dharchula wassignificantly rich in linalool (14.2%) and methyl chavi-col (78.3%) (group IV). Oils from Almora and Kilbury(group V) showed 1,8-cineole (11.5–28.4%), camphor

(49.4–42%), eugenol (0–8.6%) and aliphatic hydrocar-bons (1.1–11.4%) as major constituents, while the oilfrom Mussoorie (group VI) was significantly rich incamphor (23.0%) and aliphatic hydrocarbons (44.1%)as major constituents. The classification of these sixgroups was done on the basis of presence or absenceof their major constituents:

• Group I: Banbasa, Rushi Nainital and Champa-wat (methyl chavicol, 1,8-cineole, (E)-γ-bisabo-lene, β-bisabolene and eugenol);

• Group II: Dhoulchina ((E)-γ-bisabolene, aliphatichydrocarbons, eugenol, β-bisabolene and methylchavicol);

• Group III: Rudrapur (eugenol and (E)-caryophyl-lene);

• Group IV: Dharchula (linalool and methyl chavi-col);

• Group V: Almora and Kilbury (aliphatic hydro-carbons, eugenol, camphor and 1,8-cineole);

• Group VI: Mussoorie (camphor and aliphatichydrocarbons).

Figure 2. Gas chromatogram of the essential oil of Ocimum americanum L. from Dhoulchina.

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Table

4.Correlatio

nmatrix(r)am

ongiron

(Fe),copp

er(Cu)

insoilandplantwith

major

constituentsin

soil.

12

34

56

78

910

1112

1314

1516

S.No.

Fe

FeDTPA

FePlant

Cu

CuDTPA

CuPlant

1,8-

Cineole

Linaloo

lCam

phor

Methy

lchavicol

Eug

enol

(E)-Caryo

-ph

yllene

β-Bisabolene

δ-Cadinene

(E)-α-

Bisabolene

Alip

hatic

hydrocarbo

ns

11.00

0.05

90.18

60.24

20.34

70.10

80.34

8�0

.383

�0.364

0.01

90.25

0�0

.217

�0.228

21.00

0.47

6�0

.132

�0.093

0.60

0�0

.263

�0.533

�0.113

�0.493

�0.232

�0.351

0.67

6⁄

31.00

0.56

2�0

.371

0.51

9�0

.451

�0.369

�0.524

�0.085

�0.267

0.07

60.50

14

1.00

0.05

20.93

9⁄⁄

0.07

80.50

20.18

30.29

3�0

.359

0.16

5�0

.547

�0.109

�0.569

0.05

45

1.00

�0.173

0.18

0�0

.166

�0.106

�0.092

0.15

0�0

.221

0.27

3�0

.280

0.40

6�0

.073

61.00

0.07

20.59

60.30

90.34

7�0

.460

0.21

5�0

.678

⁄�0

.015

�0.747

⁄�0

.010

71.00

�0.219

0.48

9�0

.343

�0.031

�0.332

�0.027

�0.280

0.15

9�0

.316

81.00

�0.022

0.91

3⁄⁄

�0.571

�0.233

�0.382

0.04

6�0

.432

�0.145

91.00

�0.372

�0.615

�0.236

�0.584

�0.144

�0.496

0.33

810

1.00

�0.366

�0.237

�0.090

0.19

1�0

.216

�0.221

111.00

0.52

40.55

8�0

.145

0.67

1⁄�0

.531

121.00

�0.299

�0.107

�0.231

�0.133

131.00

0.33

90.78

8⁄⁄

�0.341

141.00

�0.294

0.05

815

1.00

�0.386

161.00

Notes:⁄ C

orrelatio

nissignificant

atthe0.05

level.

⁄⁄Correlatio

nissignificant

atthe0.01

level.

Table

5.Correlatio

nmatrix(r)betweenmicroclim

atic

cond

ition

sandmajor

constituentsof

essentialoil.

12

34

56

78

910

1112

1314

Tem

pAltitude

Oil%

Plant

height

1,8-

Cineole

Linaloo

lCam

phor

Methy

lchavicol

Eug

enol

(E)-

Caryo

phyllene

β-Bisabolene

δ-Cadinene

(E)-α-

Bisabolene

Alip

hatic

hydrocarbo

ns

11.00

�0.888

⁄⁄�0

.516

�0.757

⁄�0

.223

�0.330

�0.099

�0.280

0.47

50.76

2⁄�0

.195

�0.040

�0.084

�0.161

21.00

0.62

90.62

90.18

70.50

30.26

30.38

9�0

.646

⁄�0

.695

⁄�0

.002

0.13

1�0

.181

0.07

53

1.00

0.51

0�0

.232

0.78

5⁄⁄�0

.319

0.83

7⁄⁄

�0.327

�0.270

0.09

70.44

8�0

.248

�0.278

41.00

0.50

00.29

50.113

0.20

0�0

.239

�0.391

0.04

80.05

1�0

.063

�0.173

Notes:⁄ C

orrelatio

nissign

ificant

atthe0.05

level.

⁄⁄Correlatio

nissignificant

atthe0.01

level.

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The main component in the volatile leaf oil constit-uents of Ocimum americanum L. collected from Wes-tern Kenya was terpinen-4-ol (43.21%), followed by1,8-cineole (16.13%) and α-terpineol (4.01%) alongwith cis-sabinene hydrate (2.59%), α-bergamotene(2.68%) and trans-caryophyllene (3.06%) as minor con-stituents (9). Mondello et al. (10) have reported thepresence of two chemotypes of O. americanum. Onebeing the citral-type oil, according to its content ofneral (27.9%) and geranial (37.7%), whereas the otheroil was characterized by a high content of camphor(38.6%). In our report, the main constituents of oilswere different from these reports. Our results confirm ahigh variation in essential oil composition and thepresence of various chemotypes in O. americanum.Essential oil composition of O. americanum wasaffected by variation in soil properties and microclimat-ic conditions.

3.2. Correlations

All the statistically significant correlation coefficientsare given in Tables 4–6. Linalool was positivelycorrelated with methyl chavicol while eugenol with(E)-α-bisabolene and β-bisabolene with (E)-γ-bisabo-lene. Correlation analysis revealed that the essential oilcomposition was affected by variation in soil micronu-trients in soil and plant. Available iron was positivelycorrelated with aliphatic hydrocarbons, while total cop-per in plant was negatively correlated with β-bisaboleneand (E)-γ-bisabolene, indicating the role of iron andcopper in their biosynthesis in O. americanum (Tables4 and 5). Kanias et al. (21) found a significant negativecorrelation of iron, scandium and chromium with carva-crol and positive with thymol in Origanum vulgare,suggesting that these correlations could make probablerole of the particular elements in the biosynthesis of thepredominant compounds. Percentage oil yield was posi-tively correlated with linalool and methyl chavicol.Temperature was positively correlated with (E)-caryo-phyllene (Table 6). Several reports on phenol rich Lam-iaceae species have shown that their essential oilcompositions were significantly associated to climaticconditions (22, 23) and Vokou et al. (22) found that thesum of phenolic compounds was positively correlatedwith air temperature. Rahimmalek et al. (24) found thathigh level of inter- and intra-species chemical polymor-phism among Achillea species was due to genetic andenvironmental factors and their interactions. In ourstudy, the variation in chemical composition does notseem to be altitudinal/climatic; rather, it may begenetic. A similar study on the chemotypic variation ofessential oils in Anemopsis californica has largelyattributed it to genetic factors (25).

Iron in soil positively affects the biosynthesis of ali-phatic hydrocarbons and copper negatively affects theTa

ble6.

Correlatio

nmatrix(r)betweenph

ysical

prop

ertiesof

soilandmajor

constituentsof

essentialoil.

12

34

56

78

910

1112

1314

1516

1718

pHEC

OC%

CEC

WHC

Sand%

Silt%

Clay%

1,8-

Cineole

Linaloo

lCam

phor

Methy

lchavicol

Eug

enol

(E)-

Caryo

-ph

yllene

β-Bisabolene

δ-Cadinene

(E)-α-

Bisabolene

Alip

hatic

hydrocarbo

ns

11.00

0.29

90.05

2�0

.036

�0.173

�0.239

0.47

4�0

.236

0.14

2�0

.427

0.22

9�0

.430

0.14

30.30

7�0

.179

0.05

9�0

.137

0.21

22

1.00

0.58

10.37

80.40

6�0

.277

0.26

30.17

1�0

.406

�0.112

0.13

4�0

.099

�0.290

�0.005

�0.129

0.14

0�0

.252

0.59

43

1.00

0.43

2�0

.297

�0.634

⁄0.50

90.44

5�0

.573

�0.238

0.02

9�0

.260

0.09

50.48

2�0

.041

0.34

3�0

.303

0.20

84

1.00

�0.179

0.18

1�0

.056

�0.364

0.15

4�0

.534

0.16

2�0

.452

0.07

5�0

.049

0.18

10.06

50.13

00.09

75

1.00

0.29

5�0

.290

�0.071

0.03

60.43

60.31

80.30

3�0

.749

⁄�0

.536

�0.379

�0.232

�0.288

0.57

46

1.00

�0.900

⁄⁄�0

.711

⁄0.73

5⁄�0

.125

0.25

0�0

.135

�0.148

�0.813

⁄⁄0.37

4�0

.185

0.48

6�0

.130

71.00

0.35

3�0

.501

�0.036

�0.001

�0.080

0.10

90.81

7⁄⁄�0

.562

�0.043

�0.491

0.21

78

1.00

�0.766

⁄⁄0.51

0�0

.505

0.60

1�0

.013

0.36

9�0

.017

0.49

5�0

.377

�0.065

Notes:EC,electrical

conductiv

ity;OC%,organiccarbon;CEC,catio

nexchange

capacity;WHC,water

holdingcapacity.⁄ C

orrelatio

nis

significant

atthe0.05

level.

⁄⁄Correlatio

nis

significant

atthe

0.01

level.

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synthesis of β-bisabolene and (E)-γ-bisabolene in O.americanum. Under warm conditions, the plant synthe-sizes more (E)-caryophyllene. In moist soils, there areless chances of the synthesis of eugenol.

AcknowledgementsThe author is grateful to the Director, USERC, Dehradun, forfinancial support and the Head, Chemistry Department,Kumaun University, Nainital, for providing the necessarylaboratory facilities and encouragement.

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Figure 6. Gas chromatogram of the essential oil of Ocimum americanum L. from Mussoorie.

Figure 7. Agglomerative hierarchical clustering analysis (Dendrogram) by SPSS 16.0 for the chemical abundances of 12essential oil components in the 10 populations of Ocimum americanum L.

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