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Chapter- 4

Effect of soil properties and microclimatic

conditions on essential oil composition of

Origanum vulgare L. and its chemosystematics

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4.1. Introduction

The genus Origanum belonging to the family Lamiaceae is indigenous to the

Mediterranean region. It is also distributed and cultivated in many areas of the mild and

temperate climates of Europe, Asia, North Africa and America1. It is characterized by a

large morphological and chemical diversity, having 49 taxa divided into 10 sections. In

particular 3 taxa are restricted to Morocco and South Spain, 2 occur in Algeria and

Tunisia, 3 endemic to Cyrenaica, 9 restricted to Greece, South Balkans and Asia

minor, 21 in Turkey, Cyprus, Syria and Lebanon and 8 locally distributed in Israel,

Jordan and Sinai Peninsula1-3

. Ecologically, the species of Origanum prefer warm,

sunny habitat and loose, often rocky, calcareous soils low in moisture content.

Origanum vulgare L. commonly known as Himalayan marjoram is widely

distributed in Eurasia and North Africa. This species has also been encountered in North

America1, 4

. It is an erect perennial aromatic herb, with small pale, pink flowers

crowded in to a branched domed inflorescence. This plant is 20-80 cm high with ovate

entire, stalked leaves 1-4 cm and flowers from May to October5. One of the

considerable morphological characteristic of the Origanum plant is the presence of

glandular and non glandular hair (peltate hair on glandular scales) covering the aerial

organ. Both types of hair originate from epidermal cells6. The glandular hair are

numerous on the vegetative organ such as stems, leaves and bracts, while their density

become reduced on the reproductive organs such as calyces and corollas7. The glandular

hair produces and secretes an essential oil with a characteristic odour, mainly due to

monoterpenes being the major components of the oil8. Six subspecies have been

recognized within O. vulgare L. based on differences in indumentums, number of

sessile glands on leaves, bracts and calyces and size and color of bracts and flowers2.

1) O. vulgare L. subsp. vulgare- Europe, Iran, India, China

2) O. vulgare L. subsp. glandulosum - Algeria, Tunisia (Desfontaines) Ietswaart

3) O. vulgare L. Subsp. gracile (Koch) - Afghanistan, Iran, Turkey, former USSR

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Ietswaart

4) O. vulgare L. subsp. hitrum (Link)- Albania, Croatia, Greece, Turkey

Ietswaart

5) O. vulgare L. subsp. viridulum - Afghanistan, China, Croatia, France, Greece,

(Martrin-Donos) Nyman India, Iran, Italy, Pakistan

6) O. vulgare L. subsp. virens - Azores, Balearicls, Canary Is,

(Hoffmannsegg & Link) Ietswaart Madeira, Morocco, Portugal, Spain

Herbal parts of Origanum species used by local people as herbal tea and spice

in soups, salads, sausages olives and meats9. They are also used for production of

essential oil and the remaining distilled water is taken orally to reduce blood cholesterol

and glucose levels and also for cancer10

. In folk medicine, it is also used as stimulant,

emmenagogic, stomachic, analgesic, antitussive, expectorant, sedative, antiparasitic and

antihelminthic11

. The volatile oil of oregano has also been used traditionally for

respiratory disorders, dental caries, rheumatoid arthritis and urinary tract disorders12

.

Carvacrol is a major active component of oregano and has potential uses as a food

preservative13

. Tepe et al. (2004) 14

suggested that the essential oil and extract from the

herbal part of O. syriacum could be used as natural preservative ingredients in the food

industry. O. vulgare L. is also used for perfumery, cosmetic preparations and aromatic

compounds of strong and non alcoholic drinks, medicines, essential oils, extracts and

waxes having biological active compounds 15

. It is also used to produce scented grape

wines16

. The essential oil of many Origanum species have been proven to possess

antibacterial17

, antifungal18

and antioxidant properties19

. Origanum vulgare L.20

, O.

compactum21

, O. majorana L.22

, O. creticum L.23

, O. syriacum L.24

and O. acutidens25

showed larvicidal activity against various insect species.

The essential oil of oregano plants is characterized by a high carvacrol content26,

27. Carvacrol is known for its antibacterial and antifungal activities, antispasmodic

effects, acetylcholine esterase inhibition, lipid peroxidase inhibition, radical scavenging

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effect, white blood cell macrophage stimulant and cardiac depressant activity28

. The

essential oil composition of O. vulgare L. has been reported from many countries5, 15

.

Origanum vulgare L. growing in ten different localities of Vilnius district (Lithuania)

has two chemotypes: ß-ocimene type and germacrene D type29

. Carvacrol has been

reported as the major component of the O. vulgare ssp. hirtum30

. Three chemotypes of

O. vulgare L. namely carvacrol/thymol, thymol/α-terpeniol and linalyl acetate/linalool

growing wild in Campania (Southern Italy) have been reported by De Martino et al.

(2009)31

. D'antuono et al. (2000)15

reported three chemotypes of O. vulgare collected

from Northern Italy: carvacrol/thymol type, (E)-caryophyllene/γ-muurolene/high

linalool type and β-bourbonene/(E)-caryophyllene/γ-muurolene/germacrene D-4-

ol/caryophyllene oxide type. Russo et al. (1998)32

reported four chemotypes for O.

vulgare growing in Calabria (Southern Italy), on the basis of their phenolic content:

thymol, carvacrol, thymol/carvacrol and carvacrol/thymol chemotypes. The essential oil

of O. vulgare from Turkey contained caryophyllene (14.4%), spathulenol (11.6%),

germacrene D (8.1%) and aterpineol (7.5%) as the main constituent33

. Cosge et al.

(2011)34

reported thymol as the major constituent in the essential oil of O. vulgare L.

ssp. hirtum. The main constituents of O. vulgare L. subsp. viride growing in Iran were

linalyl acetate, β-caryophyllene and sabinene while the O. vulgare subsp. virens plants

produce linalool, β-caryophyllene, linalool/α-terpineol, linalool/terpinen-4-ol, terpineol

(-linalool) and terpineol (-carvacrol) chemotypes35

. Considering huge genetic diversity,

the information on O. vulgare from India is scanty36

.

The essential oil compositions of medicinal and aromatic plants are not constant

but vary quantitatively and qualitatively. Essential oil quality depends upon different

environmental factors like nature of soil, climatic conditions like light, temperature,

altitude, moisture, growing and harvesting time etc37, 38

. Origanum vulgare growing in a

Mediterranean climate or a continental one contains a higher amount of phenols39

or

terpenic alcohols40

. However, the variability between commercial and wild plant

growing under the same climatic condition remains high39

. The variation of the

chemotypes has also been stated by Burkart and Buhler (1997)41

as the result of

interaction between the type of aromatic vegetation and several environmental factors.

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Kokkini et al. (1997)42

studied the essential oils from O. vulgare ssp. hirtum plants

collected in late autumn from six localities of three distinct geographic areas of Greece.

They reported that oils of plants from the Northern part of Greece were rich in thymol,

whereas those from the Southern part of country were rich in carvacrol. A wide

chemical diversity is found even within a single Origanum species i.e. the widely used

O. vulgare where the pattern of variation of quantitative and qualitative essential oils

depends on geographical distribution or on the time of plant collection3. One important

factor, which affects the essential oil composition of aromatic and medicinal plants, is

the content of macro and micronutrients in soil as well as in plants. They play a very

important role in the biogenetic pathways of different secondary metabolites of oil.

Chemical fertilization, particularly nitrogen (N), strongly affected not only herb yield

but also its essential oil content and major oil constituents. According to Omer (1999)43

nitrogen fertilization on O. syriacum L. seemed to increase the biosynthesis of thymol

and carvacrol with the decrease in the content of α-terpinene and p-cymene. The

desirable amount of carvacrol, p-cymol and γ-terpinene were obtained at 40 and 60 kg

ha-1

nitrogen application44

. Effect of nitrogen fertilization on essential oil of O. vulgare

L. has been reported by Said-Al Ahl et al. (2009)45

. An increase in the percentage of p-

cymene accompanied by a decrease in the percentage of carvacrol observed when

phosphorus was used in nutrient solution, especially in the case of leaves of O.

dictamnus46

. Kanias et al. (1998)47

reported that iron, chromium and scandium showed

a negative significant correlation with carvacrol and positive with thymol. It is

necessary to study the relationship among the metal content in soil, plant, environmental

factors and active constituents of aromatic and medicinal plants to examine which metal

or trace element or environmental factor is responsible for any increased or decreased

variation of active constituents of aromatic and medicinal plant.

To the best of our knowledge, no work based on the chemosystematics of O.

vulgare with respect to different soil and geographic conditions has been undertaken in

Uttarakhand. Therefore, the objective of the present investigation is to explore the

chemosystematics of this important genus with respect to microclimatic conditions in

Uttarakhand, India.

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4.2. Collection of plant material and soil samples

Fresh plant material of O. vulgare L. along with its soil samples (0-20 cm) were

collected in September to November, 2009 from ten locations viz. Dhoulchina

(29°37'N, 79º40'E), Champawat (29°36'N: 79°30'E), Dharchula (29°51'00''N:

80°31'60''E), Munsiyari (30°04'37"N: 80°23'04"E), Ramgarh (29º23'N: 79º30'E),

Kilbury (29º23'N: 79º30'E), Mukteshwar (29°28'N: 79°39'E), Mussoorie (30º 27' N: 78º

06' E), Nainital (29º23'N: 79º30'E) and Rushi village (29º23'N: 79º30'E) in Kumaun

Himalaya (Uttarakhand, India). The plants were in full blooming stage. The botanical

identification of the specimen was done at Botany Department, Kumaun University,

Nainital and deposited at Botanical Survey of India, Dehradun (Voucher no. -2036).

4.3. Fractionation of the oil and identification of major

compounds

The essential oils of O. vulgare L. (5.0 mL) were fractionated by using column

chromatography (CC) on a column packed with 100 g silica gel (230-400 mesh) in n-

hexane (Scheme 4.1 and 4.2). The fractions (OV # 1 and OV # 2) obtained by column

chromatography were analyzed by spectroscopy (1H and

13C NMR) and MS to

determine their identity.

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4.3.1. Flow sheet (1) for CC of essential oil O. vulgare L.

Essential oil (5.0 mL)

n-hexane 5% Et2O 10% Et2O 15% Et2O 20 % Et2O

in n-hexane in n-hexane in n-hexane in n-hexane

Fr (1-12) Fr (12-20) Fr (21-26) Fr (27-33) Fr (33-43)

A B C D E

Recolumn 5% Et2O

OV # 01

Scheme 4.1 Isolation of compound from O. vulgare L. from Dharchula.

4.3.2. Flow sheet (2) for CC of essential oil Origanum vulgare L.

Essential oil (5.0 mL)

n-hexane 5% Et2O 10% Et2O 15% Et2O 20 % Et2O

in n-hexane in n-hexane in n-hexane in n-hexane

Fr (1-12) Fr (12-20) Fr (21-26) Fr (27-33) Fr (33-43)

A B C D E

Recolumn 5% Et2O

OV # 02

Scheme 4.2 Isolation of compound from O. vulgare L. from Rushi village.

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4.4. Results and Discussion

4.4.1. Characterization of the constituents

1) Characterization of OV#01:

Physico-chemical data

IR vmax cm-1

(Figure 4.1) : 3425, 2963, 2871, 1708, 1623, 1584, 1348, 831, 770

EIMS (70eV): 150 (M+), 135 (100%), 115, 107, 105, 91, 77.

1H NMR (300MHz, CDCl3-TMS) (Figure 4.2):

δ 1.15-1.17(d, 6H), 2.19 (s, 3H), 3.06-3.10 (sept, 1H), 4.56 (1H, ArOH), 6.50 (s, 1H),

6.64-6.66 (d, 1H), 6.98-7.01 (d, 1H).

13CNMR (75MHz, CDCl3-TMS) (Figure 4.3):

δ 136.3 (s, C-1), 126.3 (d, C-2), 152.9 (s, C-3), 131.8 (s, C-4), 121.7(d, C-5), 116.1 (d,

C-6), 20.9 (q, C-7), 26.8 (d, C-8), 22.7 (q, C-9), 22.7 (q, C-10).

The compound OV#1 isolated, was a dark yellow liquid. The EIMS of the

compound showed a molecular ion peak at m/z 150 corresponding to the molecular

formula C10H14O. Considering the unsaturation in the molecular formula, the compound

must be monocyclic with aromatic nucleus in the molecule as is evident by the 1H

NMR signals in between δ 6.50- 7.01 (4H). This corresponds to hydrogen deficiency of

four. 13

CNMR spectra shows 10 carbon resonances which were attributed to three

methyl, four methylene and three quaternary carbon atoms by DEPT assignment. Based

on these spectral data, OV#1 has been identified as thymol. Finally, its identity was

confirmed by comparison of its spectral data with those reported in literatures48, 49

.

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Figure 4.1 IR Spectrum of OV #1

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Figure 4.2 1H NMR Spectrum of OV #1

Figure 4.3 13

C NMR Spectrum of OV #1

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2) Characterization of OV#02:

Physico-chemical data

IR vmax cm-1

(Figure 4.4): 2925, 2850, 1735, 1480, 1380, 1245, 910 .

EIMS (70eV): 196 (M+), 154, 136, 121, 108, 95, 93, 91, 80, 67.

1H NMR (300MHz, CDCl3-TMS) (Figure 4.5):

δ 0.93 (6 H, s, Me-9/10), 1.28 (3 H, s, Me-7), 2.06 (3 H, s, Me-12), 4.89 (1 H, m,

H-1).

13CNMR (75MHz, CDCl3-TMS) (Figure 4.6):

δ 79.8 (d, C-1), 48.6 (s, C-2), 36.7 (t, C-3), 27.0 (t, C-4), 44.8 (d, C-5), 28.0 (t,

C-6), 22.3 (q, C-7), 47.7 (s, C-8), 19.8 (q, C-9), 13.4 (q, C-10), 171.4 (s, C-11), 20.5(q,

C-12).

The compound OV#2 isolated was obtained as a viscous liquid. The EIMS of the

compound displayed a molecular ion peak at m/z 196 [M+] corresponding to the

molecular C12H20O2 with another fragment peak at m/z 154 [M+] suggesting that the

compound could be a monoterpene ester. The methane carbon (C-1) directly attached to

oxygen atom of ester group (O-C=O) resonate at δ 79.8 (d). The signal for carbonyl

carbon of the ester group appeared at δ 171.4 and the adjacent methyl hydrogens

resonate at δ 2.06 (3H, s). A total of 12 carbons appear in the 13

C NMR of the

compound. Based on these spectral data, OV#2 has been identified as bornyl acetate.

Finally, its identity was confirmed by comparison of its spectral data with those

reported in literatures48, 50

.

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Figure 4.4 IR Spectrum of OV#2

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Figure 4.5 1H NMR Spectrum of OV#2

Figure 4.6 13

C NMR Spectrum of OV#2

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4.4.2. Chemosystematics of O. vulgare L.

The structures of major constituents are shown in Figure 4.7. The essential oils

of O. vulgare L. collected from ten sites were analyzed by cluster analysis (Figure 4.8).

The result of cluster analysis grouped these essential oils into four clusters on the basis

of difference in their chemical constituents and allowing them to be characterized into

four distinct chemotypes (Table 4.1). Mukteshwar (Figure 4.9), Rushi village (Figure

4.10), Mussoorie (Figure 4.11), Nainital (Figure 4.12) and Kilbury (Figure 4.13)

(chemotype I) showed bicyclogermacrene (0.3-5.4%), elemol (0.8-5.3%), α-Cadinol

(1.1-8.8%), linalool (5.1-9.7%), germacrene D (6.3-18.0%), bornyl acetate (6.9-18.6%)

and (E)-caryophyllene (9.2-16.7%) as the major constituents. The oil from Ramgarh

(Figure 4.14) (chemotype II) represents thymol (5.1%), germacrene D (5.7%),

carvacrol (7.5%), α-Cadinol (9.3%) (E)-caryophyllene (10.4%) and linalool (10.9%) as

major constituents. The oil of O. vulgare L. collected from Dhoulchina (Figure 4.15)

and Champawat (Figure 4.16) (chemotype III) showed p-cymene (6.7-9.8%), -

terpinene (12.4-14.0%), carvacrol (12.4-20.9%) and thymol (29.7-35.1%) as the major

constituents while oil of from Dharchula (Figure 4.17) and Munsiyari (Figure 4.18)

(chemotype IV) showed the presence of caryophyllene oxide (7.5-7.6%), aliphatic

hydrocarbons (12.8-35.1%) and thymol (30.2-55.1%).

Three chemotypes of O. vulgare L. were observed by De Martino et al. (2009)36

:

carvacrol/thymol (21.9%/18.2%); thymol/α-terpineol (26.8%/15.1%) and linalyl

acetate/linalool (15.9%/12.5%) type.

The cluster analysis result grouped the essential oils into four chemotypes on the

basis of presence or absence of chemical markers.

Chemotype I: Bicyclogermacrene, elemol, α-Cadinol, linalool, germacrene D, bornyl

acetate and and (E)-caryophyllene.

Chemotype II: Thymol, germacrene D, carvacrol, α-Cadinol, (E)-caryophyllene and

linalool.

Chemotype III: p-Cymene, -terpinene, carvacrol and thymol.

Chemotype IV: Caryophyllene oxide, aliphatic hydrocarbons and thymol.

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The chemical composition of essential oils isolated from aerial parts of O.

vulgare from Northwestern Himalaya was investigated by Bisht et al. (2009)40

. They

have reported the presence of linalool (11.0-14.7 %), bornyl acetate (7.0-9.3 %) along

with borneol (3.4-5.9 %), terpinen-4-ol (0.6-1.2 %) and α-terpineol (2.4-8.4 %) in

Nainital and Bhowali regions. Our reports are similar to the chemical composition of

essential oils isolated from aerial parts of O. vulgare from Northwestern Himalaya by

Bisht et al. (2009)40

. The only significant difference is the presence of aliphatic

hydrocarbons (12.8 to 35.1%) along with thymol and caryophyllene oxide in

Chemotype IV.

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OH

para-cymene (12) ץ-terpinene (17) linalool (19)

MF - C10H14 MF-C10H16 MF-C10H18O

FW – 134 g/mol FW-136 g/mol FW-154 g/mol

O

O

OH

OH

bornyl acetate (31) thymol (32) carvacrol (33)

MF-C12H20O2 MF-C10H14O MF- C10H14O

FW-196 g/mol FW-150 g/mol FW-150 g/mol

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(R)

(S)

(R)

(E)-caryophyllene (41) germacrene D (44) bicyclogermacrene (47)

MF- C15H24 MF- C15H24 MF- C15H24

FW- 204 g/mol FW- 204 g/mol FW-204 g/mol

OH O

β-bisabolene (49) elemol (51) caryophyllene oxide (55)

MF- C15H24 MF- C15H26O MF- C15H24O

FW-204 g/mol FW-222 g/mol FW-220 g/mol

Figure 4.7 Structure of major compounds

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Figure-4.8 Agglomerative hierarchical clustering analysis (Dendrogram) by

SPSS 16.0 for the chemical abundances of 13 essential oil components in the

10 populations of O. vulgare L.

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Figure 4.9 GC of the essential oil of O. vulgare L. collected from Mukteshwar

Figure 4.10 GC of the essential oil of O. vulgare L. collected from Rushi village

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Figure 4.11 GC of the essential oil of O. vulgare L. collected from Mussoorie

Figure 4.12 GC of the essential oil of O. vulgare L. collected from Nainital

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Figure 4.13 GC of the essential oil of O. vulgare L. collected from Kilbury

Figure 4.14 GC of the essential oil of O. vulgare L. collected from Ramgarh

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Figure 4.15 GC of the essential oil of O. vulgare L. collected from Dhoulchina.

Figure 4.16 GC of the essential oil of O. vulgare L. collected from Champawat.

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Figure 4.17 GC of the essential oil of O. vulgare L. collected from Dharchula

Figure 4.18 GC of the essential oil of O. vulgare L. collected from Munsiyari

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Table- 4.1 Chemotypes of O. vulgare collected from different sites

S.No.

Compoundsa

RIb

RIc

Chemotype I Chemot

ype II

Chemotype III Chemotype II

Muktesh

war (7)

Rushi

village (8)

Mussoori

e (9)

Nainital

(10)

Kilbury

(6)

Ramgarh

(5)

Dhoulchi

na (1)

Champa

wat (2)

Dharchul

a (3)

Munsiyar

i (4)

1 santolinatriene AH 908 906 0.3 0.4 - - - - - - - -

2 α-thujene MH 930 924 - - - - - 0.5 - 1.0 - -

3 α-pinene MH 939 932 0.7 0.9 - - 0.2 0.6 t 0.4 - -

4 camphene MH 954 946 0.9 - - - 0.6 - - - - -

5 sabinene MH 975 969 0.4 0.6 - - 1.7 3.0 t 1.5 0.3 0.8

6 β-pinene MH 979 974 - 1.9 - 2.7 - t - - 0.2

7 3-octanone AK 983 979 0.8 0.4 - - 0.4 - - 0.7 0.3 -

8 β-myrcene MH 990 988 1.9 1.1 0.6 - 0.3 - 1.3 2.3 - 0.2

9 3-octanol AA 991 988 0.2 0.4 - - 3.7 - - 0.3 - -

10 α-phellendrene MH 1002 1002 - - - - - 0.4 0.2 1.0 -

11 α-terpinene MH 1017 1014 0.3 0.2 - - 0.2 - 2.0 2.2 - 0.2

12 p-cymene MH 1024 1020 0.3 0.5 - - 0.9 - 6.7 9.8 - t

13 limonene MH 1029 1024 4.3 3.9 0.5 1.1 0.5 0.7 0.3 - - 0.2

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14 1,8-cineole OM 1031 1026 - - 4.5 3.4 2.6 1.1 0.2 0.9 -

15 (Z)- β-ocimene MH 1037 1032 - 1.3 - 4.9 1.5 - 1.0 - -

16 (E)- β-ocimene MH 1050 1044 0.6 0.6 - - - - - - - t

17 γ-terpinene MH 1059 1054 3.0 3.6 1.4 4.0 0.6 1.5 12.4 14.0 0.3 -

18 (Z)-sabinene

hydrate

MH 1070 1065 4.0 3.2 - - 2.1 - - 0. 8 0.8 0.5

19 linalool OM 1096 1095 8.5 6.7 5.1 7.7 9. 7 10.9 4.0 1.3 t 0.2

20 3-octanolacetate 1123 1120 0.9 0.3 - - - - - - - -

21 camphor OM 1146 1141 - - - - - - - - 1.4 0.4

22 n.i. - - 0.3 0.6 - - - - - 0.3 - -

23 borneol OM 1169 1165 2.4 2.9 1.5 5.4 1.2 - - 0.7 0.4 0.4

24 terpinen-4-ol OM 1177 1174 0.8 0.4 - - 1.8 - 0.6 - - 0.6

25 methyl chavicol OM 1196 1195 - - - - - - - - - 0.3

26 α-terpineol OM 1188 1186 - - 0.6 4.3 1.4 3.1 0.2 0.3 0.4 -

27 thymolmethyl

ether

OM 1236 1232 - - - - - - 0.2 3.1 0.3 0.9

28 carvacrol methyl

ether

OM 1244 1241 - - - - - - 4.5 - t 0.4

29 cumin aldehyde OM 1245 - - - - - - - - - - 0.4

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30 thymoquinone OM 1252 1248 - - - - - - - - 5.6 2.8

31 bornyl acetate OM 1288 1287 18.6 16.8 6.9 9.4 12. 6 - - - - -

32 thymol OM 1290 1289 - - - - - 5.1 29.7 35.1 55.1 30.2

33 carvacrol OM 1299 1298 - - - - - 7.5 20.9 12.4 1.9 1.0

34 δ-elemene SH 1338 1335 - - - - - - 0.3 - -

35 trans-

carvylacetate

OM 1342 1339 - 9.2 - -

36 thymylacetate OM 1352 1349 - - - - - - t - - -

37 α-copaene SH 1376 1374 1.7 1.3 - - 1.1 - - - - 1.6

38 β-bourbonene SH 1388 1387 - - 1.8 2.6 1.5 3.1 - - - -

39 β-elemene SH 1389 1388 - 2.2 1.9 3.2

40 (Z)-α-

bergamotene

SH 1412 1411 - 1.0 - - - - - 3.0 - -

41 (E)-

caryophyllene

SH 1419 1417 14.3 10.9 9.2 16.7 13.8 10.4 2.3 1.2 - 0.2

42 aromadendrene SH 1441 1439 - 0.6 - - -

43 α-humulene SH 1454 1452 0.6 0.9 - 2.8 2.0 1.7 t - - -

44 germacrene D SH 1485 1484 13.0 11.3 18.0 11.7 6.3 5.7 1.9 - - 0.7

45 δ-selinene SH 1492 1492 0.4 1.3 - - - - - - - -

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46 epi-cubebol OS 1494 1493 - - - - - - 0.4 - - -

47 bicyclogermacre

e

SH 1500 1500 0.6 0.8 2.3 5.4 0.3 3.4

48 (E),(E)-α-

farnesene

SH 1505 1505 - 1.2 1.1 0.4 2.3 - - - 0.5

49 β-bisabolene SH 1505 1505 5.3 4.7 2.1 3.9 3.2 - - - - -

50 δ-cadinene SH 1523 1522 - - - - 3.1 - 2.8 - - -

51 elemol OS 1549 1548 3.9 4.1 5.3 0.8 1.0 - - - - -

52 cis-muurol-5-en-

4-2-ol

OS 1561 1559 - - - - 2.1 - - - - -

53 germacrene D-4-

ol

OS 1575 1574 - - - - - - t 2.5 - -

54 spathulenol OS 1578 1577 1.6 2.9 - - 4.5 - - 0.5 - 1.2

55 caryophyllene

oxide

OS 1583 1582 1.4 2.6 3.7 1.8 0.9 1.0 0.4 - 7.6 7.5

56 globeulol OS 1590 1590 - 1.0 - -

57 10-epi-γ-

eudesmol

OS 1623 1622 - - 1.1 - - - 0.2 - - -

58 epi-α-cadinol OS 1640 1638 0.7 1.3 - - - - - - - -

59 α-muurolol OS 1646 1644 - 3.8 1.2 4.5

60 cubenol OS 1646 1645 - 0.7 1.7 -

61 β-eudesmol OS 1649 1650 - 0.9 - -

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127

62 α-cadinol OS 1654 1652 3.1 2.7 8.8 3.5 1.1 9.3 - - - -

63 α-bisabolol OS 1685 1685 1.3 1.2 - - 1.4 1.4 0.4 - - -

64 n-octadecane AH 1800 1800 - 0.6 0.3 0.8

65 n-nonadecane AH 1900 1900 - 0.4 - -

66 tricosane AH 2300 2300 - - - - - - - - - 4.7

67 tetracosane AH 2400 2400 - - - - - - - - 3.8 6.6

68 hexacosane AH 2600 2600 - - - - - - - - 2.7 8.0

69 heptacosane AH 2700 2700 - - - - - - - - 2.8 7.8

70 octacosane AH 2800 2800 - - - - - - - - 3.5 8.0

Percent of oil identified (%) 97.1 91.7 92.6 91.1 91.2 83.8 91.2 93.0 94.8 91.1

aMode of identification: Retention Index, coinjection with standards/Peak enrichment with known oil constituents,

bRetention indices determined on

the Equity-5 column using an n-alkane homologous series (C9–C24); cretention indices from the literature (Adams, 2007), Bold type indicates major

components, %), n.i.= not identified, AK= aliphatic ketone, AA= aliphatic alcohol, MH= monoterpene hydrocarbon, OM= oxygenated monoterpene,

SH= sesquiterpene hydrocarbon, OS= oxygenated sesquiterpene ;within the brackets, numbers denotes the accession number in dendrogram in Figure

4.8.

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128

4.4.3. Physicochemical properties of soil

Physicochemical properties of the soil are given in Table 4.2. Soils were classified as loamy

sand and sandy loam. Soils were acidic to neutral (pH 5.31 to 7.41). Most of the soil EC, OC %,

CEC and WHC values are within the limits. The content of macro and micronutrients in soil falls

within the permissible limits

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129

Table 4.2 Physicochemical properties of soil used in the study

General soil properties Mukteshwar

Rushi

Mussoorie Nainital Kilbury Ramgarh Dhoulchina Champawat Dharchula Munsiyari

Mechanical

analysis

Sand (%) 76 82 68 78 84 70 66 82 66 70

Silt (%) 18 16 26 12 14 22 20 16 22 26

Clay (%) 6 4 6 8 2 8 16 2 16 8

Texture Loamy sand Loamy sand Sandy loam Loamy sand Loamy sand Sandy loam Sandy loam Loamy sand Sandy loam Sandy loam

Other soil

properties

pH (1:2)

7.41±0.10

6.22±0.61

7.40±0.11

5.60±0.61

7.32±0.11

6.62±1.00

5.81±0.20

6.30±0.30

5.31±0.40

5.41±0.20

O.C. % 2.65±0.35 3.90±0.61 3.00±0.70 3.23±0.07 1.21±0.30 3.15±0.04 1.70±0.40 0.60±0.30 4.10±0.71 4.20±0.40

EC 0.34±0.02 0.10±0.00 0.18±0.01 0.07±0.00 0.10±0.00 0.19±0.01 0.12±0.02 0.05 0.20±0.00 0.11±0.02

CEC 38.11±0.30 16.31±1.21 18.11±0.21 25.83±0.51 19.72±0.20 15.12±1.38 10.31±0.29 27.36±0.79 10.30±0.20 27.30±2.01

WHC 46.10±1.50 38.21±1.65 58.31±0.60 39.61±1.56 49.71±2.19 35.10±0.19 37.87±2.26 39.69±0.47 56.00±2.50 54.59±1.68

Total

content

(mg kg-1

)

Zn

91.680±0.43

26.880±1.17

54.280±0.57

40.500±2.13

45.280±1.42

35.280±0.67

32.150±0.48

43.620±0.63

35.050±0.91

38.670±0.08

Fe 559.250±2.94

516.860±4.09

522.060±1.79

536.480±0.49

528.870±6.99 526.470±8.85

543.640±14.00

564.450±12.87

525.630±5.03

519.330±1.03

Mn 348.020±1.42

84.150±1.82

220.900±0.59

264.400±0.59

252.010±0.62 219.350±0.04

194.620±0.50 185.810±2.18

160.100±1.63

165.000±0.85

Cu 15.550±0.02 10.700±1.75 19.830±0.88 15.300±0.59 34.680±0.27 10.830±0.88 15.500±1.15 12.900±0.71 23.400±0.35 24.630±0.68

Available

content

(mg kg-1

)

Zn 10.260±0.67 0.650±0.24 4.980±2.35 7.370±0.99 9.450±1.18 0.800±0.40 0.610±0.32 1.240±0.28 0.780±0.05 0.950±0.32

Fe 57.980±0.37 29.000±0.04 91.700±1.39 28.770±2.42 29.940±0.81 33.280±0.71 22.650±1.98 35.380±0.55 36.680±2.35 30.000±0.95

Mn 17.400±0.08 3.000±0.91 17.110±0.42 15.430±0.42 14.560±0.51 10.000±0.11 7.080±0.43 6.680±0.23 14.280±0.48 6.200±0.63

Cu 7.330±0.25 0.320±0.02 1.820±0.01 1.830±0.60 2.260±0.01 0.400±0.28 0.150±0.09 0.530±0.50 1.120±0.01 0.900±0.06

Macro

Nutrients

(%)

N (av) 0.0138±0.03 0.012±0.67 0.012±0.29 0.009±0.25 0.011±0.09 0.012±1.54 0.005±1.34 0.009±0.78 0.004±0.01 0.008±0.50

N (tot) 0.350±0.03 0.240±0.05 0.250±0.01 0.180±0.02 0.210±0.01 0.300±0.03 0.200±0.00 0.280±0.01 0.300±0.05 0.280±0.06

P(av) 0.0024±0.00 0.0007±0.00 0.0009±0.00 0.0014±0.00 0.0037±0.001 0.0033±0.00 0.0026±0.00 0.0009±0.00 0.0030±0.00 0.0011±0.00

K(av) 0.010±0.001 0.00±0.002 0.02±0.001 0.01±0.001 0.03±0.010 0.020±0.010 0.02±0.020 0.020±0.001 0.24±0.030 0.02±0.010

*(av)=Available, (tot)= Total, EC= Electrical conductivity (dS cm-1) WHC= Water holding capacity, CEC= Cation exchange capacity (c mol kg-1), O.C.%= Organic carbon %

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130

4.4.4 Microclimatic conditions and oil properties

Microclimatic conditions and oil properties are shown in Table 4.3.

Table 4.3 Microclimatic conditions and oil properties

Properties Mukteshwar Rushi Mussoorie Nainital Kilbury Ramgarh Dhoulchina Champawat Dharchula Munsiyari

Altitude (m) 2286 1600 2333 2100 2134 1789 1800 1650 2183 2235

Temperature

(0C)

18 28 20 22 23 23 24 26 22 25

Plant height

(inch)

12.23±0.306 15.97±0.76

3 16.07±0.208

15.67±0.

306

13.96±0.80

2

13.10±0.55

6 11.87±0.351 13.27±0.404 11.60±0.656 8.10±1.053

Sun/Shady

side

Shady Shady Sunny Sunny Sunny Shady Shady Sunny Sunny Sunny

Month of

collection in

2009

November November September Novemb

er September November October September October October

Oil colour Colourless Colourless Colourless Colourle

ss Colourless Light

yellow Dark yellow Dark yellow Dark yellow Dark yellow

Oil yield (%) 0.52 0.41 0.4 0.41 0.40 0.60 0.62 0.53 1.44 1.73

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4.4.5. Correlation among major constituents

The statistically significant correlations within the major constituents of O.

vulgare are given in Table 4.4. As shown in the table, there is considerable number of

significant correlations. p-Cymene is positively correlated with γ -terpinene and

carvacrol (r=0.950, P≤0.01; r=0.812, P≤01, respectively). γ-Terpinene was found to be

positively correlated with carvacrol (r=0.826, P≤0.01). Poulose and Croteau (1978)51

have reported that γ-terpinene and p-cymene are the biosynthetic precursors (via

enzymatic hydroxylation) of the two isomeric phenols, carvacrol and thymol, in Thymus

vulgaris essential oil. The variation of the concentration of thymol and carvacrol

depends upon monoterpene hydrocarbons. In addition, some monoterpene hydrocarbons

as a total (β-pinene, myrcene, camphene, α-terpinene, γ-terpinene and p-cymene) were

found to be responsible for the variation of the concentration of linalool47

. In our study,

p-cymene and γ-terpinene provide evidence that these monoterpenic hydrocarbons can

be the biosynthetic precursor of carvacrol. Linalool is positively correlated with (E)-

caryophyllene (r=0.883, P≤0.01) while negatively correlated with thymol (r= -0.851,

P≤0.01) and aliphatic hydrocarbons (r= -0.641, P≤0.05). Bornyl acetate in the plant

essential oil was positively correlated with (E)-caryophyllene (r=0.775, P≤0.01),

germacrene D (r=0.707, P≤0.05), β-bisabolene (r=0.949, P≤0.01), elemol (r=0.717,

P≤0.05), and negatively correlated with thymol (r=-0.729, P≤0.05). Thymol was

negatively correlated with (E)-caryophyllene, (r=-0.908, P≤0.01), germacrene D (r=-

0.829, P≤0.01) and β-bisabolene (r=-0.786, P≤0.01). (E)-Caryophyllene was positively

correlated with germacrene D (r=0.763, P≤.05) and β-bisabolene (r=0.810, P≤0.01). All

the three components (E)-caryophyllene, germacrene D and β-bisabolene have similar

biosynthetic pathways and common precursor (E,E)-FPP52

. Germacrene D was showing

positive correlation with β-bisabolene (r=0.874, P≤0.01) and elemol (r=0.879, P≤0.01).

β-Bisabolene was positively correlated with elemol (r=0.825, P≤0.01) while

caryophyllene oxide showed positive correlation with aliphatic hydrocarbons (r=0.877,

P≤0.01).

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132

4.4.6. Effect of macronutrient on essential oil composition

Simple correlation coefficients (r) shown in Table 4.5 suggested that available

nitrogen is positively correlated with linalool (r=0.672, P≤0.05), bornyl acetate

(r=0.642, P≤0.05), (E)-caryophyllene (r=0.684, P≤0.05), germacrene D (r=0.674,

P≤0.05) and β-bisabolene (r=0.649, P ≤0.05) while negatively correlated with thymol

(r= -0.842, P≤0.01). In previous study, Arabaci et al. (2007)53

reported that nitrogen

fertilizer increased the linalool content in the essential oil of Lavandula hybrid which

also favors our result in natural conditions. Omer (1999)43

reported that nitrogen

fertilization increased the biosynthesis of thymol and carvacrol while in our finding

natural nitrogen concentration is negatively correlated with thymol. Available K2O was

found positively correlated with thymol (r=0.709, P≤0.05) and caryophyllene oxide

(r=0.642, P≤0.05).

4.4.7. Effect of micronutrient on essential oil composition

4.4.7.1. Effect of zinc (Zn)

Simple correlation (r) matrix for zinc and essential oil composition was shown

in Table 4.6. Available Zn is positively correlated with β-bisabolene (r=0.644, P≤0.05).

Kanias, et al. (1998)47

found that chromium, iron and zinc are responsible for variance

of the concentration of thymol, carvacrol and δ-cadinene. Zinc is an essential

micronutrient for plants by acting either as a metal component of various enzymes or as

a functional, structural, or regulatory cofactor associated with saccharide metabolism,

photosynthesis and protein synthesis54

. Carbon dioxide and glucose are the main

precursors of monoterpene biosynthesis. Saccharides are also a source of energy and

reducing power for terpenoid synthesis. As zinc is involved in photosynthesis and

saccharide metabolism, and as CO2 and glucose is the most likely sources of carbon

utilized in terpene biosynthesis, the role of zinc becomes very important in the terpenoid

biosynthesis55

.

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133

4.4.7.2. Effect of iron (Fe)

Simple correlation coefficients (r) are shown in Table 4.7. Total iron (Fe)

present in soil was found to be positively correlated with total iron in plant (r=829,

P≤0.05), p-cymene (r=0.693, P≤0.05) and γ-terpinene (r=0.736, P≤0.05). Kanias et al.

(1998)47

found the similar correlation (positive correlation between iron and carvacrol)

in O. vulgare collected from Greece. Available Iron is positively correlated with

germacrene D (r=0.636, P≤0.05) and elemol (r=0.759, P≤0.05). Biosynthesis of

secondary metabolites is not only controlled genetically but it also strongly affected by

environmental factors56

. Marschner (1995)54

reported that iron play a very important

role in plant metabolism. It activates catalase enzymes associated with superoxide

dismutase, photorespiration and the glycolate pathway.

4.4.7.3. Effect of manganese (Mn)

Simple correlation coefficients (r) were shown in Table 4.8. Total Mn in plant is

positively correlated with (E)-caryophyllene (r=0.659, P≤0.05) and β-bisabolene

(r=0.656, P≤0.05). Duarte et al. (2010)57

suggested that γ-cadinene, limonene, and

caryophyllene oxide have a strong relationship with micronutrient balance in soils (Zn,

Cu, Fe, Mn) in Eugenia dysenterica.

4.4.7.4. Effect of copper (Cu)

No correlation was found (Table 4.9).

4.4.8. Effect of microclimatic conditions on essential oil composition

Simple correlation coefficients (r) were shown in Table 4.10. Vokou et al.

(1993)58

reported that altitude play important role in influencing the oil content; high

values were recorded at low altitudes and the sum of the four major oil constituents,

representing the phenol pathway, seems influenced by climate thermal efficiency.

According to Said-Al Ahl et al. (2009)45

, the phenolic compounds increases in hot

season at the expense of their preceding precursors. This may be attributed to the effect

of environmental factors especially non-endaphic factors, since these plants grew in

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134

summer month under high temperature and received more solar energy then those

grown in the spring summers. These conditions accelerate the transformation of terpene

and p-cymene to phenolic compounds. But in our study there is no correlation found

between temperature and major constituents. Stage of plant appears to play an important

role as the oil percentage is concerned. Normally plants having 30 cm or 1 foot height

possess higher oil content. Plant height is positively correlated with (E)-caryophyllene

(r= -0.644, P≤0.05) and germacrene D (r=0.704, P≤0.05) while negatively correlated

with aliphatic hydrocarbons (r=-0.793, P≤0.01).

4.4.9. Effect of soil physical properties on essential oil composition

Simple correlation coefficients (r) were shown in Table 4.11. Soil pH was found

to be negatively correlated with thymol (r=-0.671, P≤0.05) and positively correlated

with elemole (r=-0.647, P≤0.05) . Percent organic carbon represent negative correlation

with p-cymene (r=-0.759, P≤0.05), γ-terpinene (r=-0.692, P≤0.05) and caryophyllene

oxide (r=-0.698, P≤0.05). Dunford and Vazquez (2005)59

demonstrated that the effect of

moisture on Maxican oregano (Lippa berlandieri schauer) and its thymol and carvacrol

composition was examined in green house test. The study showed the amount of water

received by the plant did not have any significant effect on thymol and carvacrol of the

oil extracted from Maxican oregano. In our study water holding capacity is positively

correlated with caryophyllene oxide (r=0.633, P≤0.05).

4.4.10. Micro , macro nutrients and microclimatic conditions

Simple correlation matrix shown in Table 4.12. Total Zn in soil was positively

correlated with available Zn in soil (r=0.732, P≤0.05) and total Zn in plant (r=0.880,

P≤0.01). Available Zn is positively correlated with total Zn in plant (r=0.685, P≤0.05).

Total Zn in soil is positively correlated with available copper (r=0.956, P≤0.01), total

Mn (r=0.814, P≤0.01), available Mn (r=0.639, P≤0.05) and total plant Mn (r=0.703,

P≤0.05). Available Zn is positively correlated with available copper (r=0.801, P≤0.01),

total Mn (r=0.829, P≤0.01), available Mn (r=0.758, P≤0.05) total plant Mn (r=0.935,

P≤0.01) and plant height (r=0.703, P≤0.05). Total Zn in plant is positively correlated

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135

with available copper (r=0.789, P≤0.01), total Mn (r=0.742, P≤0.05), available Mn

(r=0.672, P≤0.05) and total plant Mn (r=0.708, P≤0.05). Total iron (Fe) present in soil

was found to be positively correlated with total iron in plant (r=0.829, P≤0.05). Total

iron in soil is negatively correlated with altitude (r=-0.868, P≤0.01) and positively

correlated with temperature (r=0.756, P≤0.05). Available iron is positively correlated

with total copper in soil (r=0.856, P≤0.01). Total iron in plant is positively correlated

with total Mn in soil (r=0.663, P≤0.05) and plant height (r=0.648, P≤0.05). Available

copper positively correlated with total Mn in soil (r=0.803, P≤0.01), available Mn

(r=0.654, P≤0.05) total plant Mn (r=-0.761, P≤0.05). Total Mn in soil is positively

correlated with plant height (r=0.657, P≤0.05). Available Mn positively correlated with

plant height (r=0.707, P≤0.05).

4.4.11. Correlation between soil physical properties and micronutrients

Soil pH is positively correlated with Zn total in soil (r=0.638, P≤0.05) shown in

Table 4.13. Soil EC is positively correlated with available Cu (r=0.672, P≤0.05) while

organic carbon is negatively correlated with total iron in soil and total iron in plant (r=-

0.698, P≤0.05, r=-0.696, P≤0.05 respectively). Water holding capacity seems to be

positively correlated with total Cu in soil (r=0.664, P≤0.05).

4.4.12. Physical properties, macronutrients and microclimatic conditions

Available nitrogen is positively correlated with pH (r=0.759, P≤0.05) and

negatively correlated with clay (r=-0.774, P≤0.01) shown in Table 4.14. Oil % is

negatively correlated with pH (r=-0.671, P≤0.05). Temperature seems to be positively

correlated with cation exchange capacity (r=0.636, P≤0.05).

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136

Table 4.4 Simple correlation matrix (r) among major constituents.

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

Table 4.5 Correlation matrix (r) between macronutrients and major constituents of essential oil

* Correlation is significant at the 0.05 level., ** Correlation is significant at the 0.01 level.

1 2 3 4 5 6 7 8 9 10 11 12 13

S.N

.

p-Cymene ץ-

Terpinene

Linalool Bornyl

acetate

Thymol Carvacro

l

(E)-Caryo

phyllene

Germacrene

D

Bicycle

germacrene

β-Bisabolene Elemol Caryophyl

lene oxide

Aliphatic

hydrocarbo

ns

1 1.00 0.950**

-0.366 -0.393 0.414 0.812**

-0.476 -0.491 -0.394 -0.444 -0.362 -0.450 -0.243

2 1.00 -0.246 -0.269 0.275 0.826**

-0.317 -0.322 -0.210 -0.291 -0.255 -0.546 -0.383

3 1.00 0.570 -0.851**

-0.217 0.883**

0.566 0.528 0.541 0.294 -0.605 -0.641*

4 1.00 -0.729* -0.580 0.775

** 0.707

* 0.110 0.949

** 0.717

* -0.269 -0.410

5 1.00 0.423 -0.908**

-0.829**

-0.545 -0.786**

-0.619 0.555 0.525

6 1.00 -0.496 -0.530 -0.260 -0.626 -0.493 -0.390 -0.208

7 1.00 0.763* 0.634 0.810

** 0.477 -0.487 -0.583

8 1.00 0.525 0.874**

0.879**

-0.362 -0.481

9 1.00 0.299 0.097 -0.259 -0.336

10 1.00 0.825**

-0.293 -0.443

11 1.00 -0.217 -0.349

12 1.00 0.877**

13 1.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

N (av)

N(total)

%

P2O5

%(av)

K2O %

(av) p-

Cymen

e

-ץ

Terpine

ne

Linalo

ol

Bornyl

acetate

Thymol Carvac

rol

(E)-

Caryo

phylle

ne

Germac

rene D

Bicycle

germacr

ene

β-

Bisabol

ene

Elemol Caryophyl

lene oxide

Aliphatic

hydrocarb

ons

1 1.00 0.270 -0.146 -0.626 -0.329 -0.275 0.672* 0.642

* -0.842

** -0.488 0.684

* 0.674

* 0.344 0.649

* 0.628 -0.424 -0.389

2 1.00 0.097 0.260 -0.117 -0.189 -0.233 -0.068 0.217 -0.167 -0.341 -0.159 -0.291 -0.160 0.140 0.378 0.245

3 1.00 0.368 -0.158 -0.218 0.323 -0.121 0.067 0.133 0.040 -0320 -0.068 -0.267 -0.381 0.021 -0.143

4 1.00 -0.158 -0.262 -0.488 -0.359 0.709* -0.089 -0.470 -0.425 -0.267 -0.383 -0.304 0.642

* 0.314

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137

Table 4.6 Correlation matrix (r) among zinc (Zn) in soil and plant with major constituents in oil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

Table 4.7 Correlation matrix (r) among iron (Fe) in soil and plant with major constituents in oil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Zn

tota

l

Zn

DTPA

Zn

Plant

p-

Cymen

e

-ץ

Terpine

ne

Linalo

ol

Bornyl

acetate

Thym

ol

Carvacro

l

(E)-

Caryo

phyllene

Germacrene

D

Bicycle

germacrene

β-

Bisabolen

e

Elemol Caryophyllen

e oxide

Aliphatic

hydrocar

bons

1 1.00 0.732* 0.880

** -0.122 -0.073 0.075 0.416 -0.335 -0.301 0.173 0.353 0.012 0.418 0.516 -0.045 -0.177

2 1.00 0.685* -0.305 -0.247 0.429 0.630 -0.609 -0.493 0.610 0.494 0.283 0.644

* 0.410 -0.211 -0.341

3 1.00 -0.136 -0.200 -0.097 0.187 -0.197 -0.424 0.033 0.205 0.019 0.228 0.337 0.118 0.059

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fe

tota

l

Fe

DTPA

Fe

Plant

p-

Cymene

-ץ

Terpinene

Linalool Bornyl

acetate

Thymol Carvacrol (E)-

Caryo

phyllene

Germacrene

D

Bicycle

germacrene

β-

Bisabolene

Elemol Caryophy

llene

oxide

Aliph

atic

hydro

carbo

ns

1 1.00 -0.011 0.829** 0.693* 0.736* -0.157 -0.034 0.121 0.449 -0.163 -0.223 -0.119 -0.094 -0.111 -0.387 -0.380

2 1.00 0.345 -0.244 -0.236 -0.027 0.180 -0.300 -0.352 0.090 0.636* 0.140 0.382 0.759* -0.055 -0.176

3 1.00 0.538 0.621 -0.059 0.040 -0.136 0.319 0.036 0.157 0.142 0.134 0.162 -0.514 -0.502

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138

Table 4.8 Correlation matrix (r) among manganese (Mn) in soil and plant with major constituents in oil

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

Table 4.9 Correlation matrix (r) among copper (Cu) in soil and plant with major constituents in oil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Mn

tota

l

Mn

DTPA

Mn

Plant

p-

Cymene

-ץ

Terpinene

Linalool Bornyl

acetate

Thymol Carvacrol (E)-

Caryo

phyllene

Germacrene

D

Bicycle

germacrene

β-

Bisabolene

Elemol Caryop

hyllene

oxide

Aliphatic

hydrocar

bons

1 1.00 0.772** 0.778** -0.118 -0.40 0.300 0.229 -0.405 -0.143 0.329 0.255 0.356 0.258 0,167 -0.204 -0.308

2 1.00 0.716* -0.420 -0.381 0.176 0.216 -0.280 -0.446 0.319 0.418 0.398 0.343 0.328 0.056 -0.237

3 1.00 -0.328 -0.258 0.424 0.618 -0.619 -0.598 0.659* 0.523 0.450 0.656* 0.392 -0.188 -0.330

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Cu

tota

l

Cu

DTPA

Cu

Plant

p-

Cymene

-ץ

Terpinene

Linalool Bornyl

acetate

Thymol Carvacrol (E)-

Caryo

phyllene

Germacrene

D

Bicycle

germacrene

β-

Bisabolene

Elemol Caryophy

llene

oxide

Aliphatic

hydrocarbo

ns

1 1.00 0.050 0.309 -0.236 -0.284 -0.061 0.021 -0.232 -0.284 0.047 0.548 0.110 0.255 0.582 -0.043 0.059

2 1.00 0.206 -0.278 -0.201 0.170 0.559 -0.375 -0.419 0.301 0.366 0.040 0.521 0.492 0.040 -0.159

3 1.00 -0.217 -0.349 0.049 0.115 -0.204 -0.274 0.087 -0.005 -0.176 0.091 -0.023 0.133 0.300

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139

Table4.10 Correlation matrix (r) between microclimatic conditions and major constituents of essential oil

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Altit

ude

Oil % Temp Plant

height

p-

Cymen

e

-ץ

Terpine

ne

Linalool Bornyl

acetate

Thymo

l

Carvac

rol

(E)-

Caryo

phyllen

e

Germac

rene D

Bicycle

germacr

ene

β-

Bisabol

ene

Elemol Caryophyl

lene oxide

Aliphatic

hydrocarb

ons

1 1.00 0.321 -0.774**

-0.217 -0.577 -0.626 -0.058 0.152 -0.077 -0.567 0.153 0.321 0.066 0.271 0.251 0.397 0.366

2 1.00 0.108 -0.835**

-0.194 -0.336 -0.692* -0.533 0.706

* -0.085 -0.693

* -0.607 -0.388 -0.583 -0.458 0.915

** 0.950

**

3 1.00 -0.033 0.393 0.345 -0.286 -0.233 0.238 0.290 -0.379 -0.471 -0.252 -0.350 -0.319 0.074 0.168

4 1.00 -0.087 0.054 0.516 0.496 -0.623 -0.223 0.644* 0.704

* 0.540 0.627 0.562 -0.583 -0.793

**

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

Table 4.11 Correlation matrix (r) between physical properties of soil and major constituents of essential oil

* Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

pH EC OC% CEC Moist

ure

conten

t

Sand Silt Clay p-

Cyme

ne

-ץ

Terpi

nene

Linalo

ol

Bornyl

acetate

Thym

ol

Carva

crol

(E)-

Cary

o

phyll

ene

Ger

macr

ene

D

Bicy

cle

germ

acre

ne

β-

Bisa

bole

ne

Elem

ol

Cary

ophy

llene

oxid

e

Alip

hatic

hydr

ocar

bons

1 1.00 0.398 -0.422 0.311 0.094 0.315 -0.061 -0.596 -0.100 -0.116 0.544 0.509 -0.671* -0.233 0.470 0.578 0.045 0.521 0.647* -0.541 -0.562

2 1.00 0.406 0.137 0.305 -0.428 0.360 0.254 -0.611 -0.524 0.218 0.259 -0.241 -0.377 0.177 0.391 0.167 0.283 0.472 0.254 -0.058

3 1.00 -0.151 0.299 -0.428 0.445 0.358 -0.759* -0.692* -0.097 0.048 0.047 -0.527 0.023 0.190 0.212 0.108 0.159 0.698* 0.542

4 1.00 0.036 0.435 -0.224 -0.540 -0.001 0.031 -0.017 0.370 -0.313 -0.348 0.180 0.180 0.093 0.347 0.253 0.006 0.077

5 1.00 -0.333 0.552 0.091 -0.392 -0.546 -0.482 -0.070 0.233 -0.508 -0.308 0.067 -0.283 0.025 0.259 0.633* 0.528

6 1.00 -0.795** -0.825** 0.151 0.130 0.406 0.614 -0.446 -0.279 0.541 0.184 0.010 0.499 0.135 -0.449 -0.369

7 1.00 0.374 -0.209 -0.304 -0.440 -0.508 0.325 0.033 -0.575 -0.100 -0.228 -0.412 0.085 0.495 0.558

8 1.00 -0.035 0.037 -0.393 -0.510 0.586 0.392 -0.480 -0.372 -0.066 -0.483 -0.369 0.441 0.269

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Table 4.12 Simple correlation matrix of micro, macronutrients and microclimatic conditions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Zn

total

Zn

DTPA

Zn

Plant

Fe

total

Fe

DTPA

Fe

Plant

Cu

total

Cu

DTPA

Cu

Plant

Mn

total

Mn

DTPA

Mn

Plant

N (av) N (t)

%

P2O5

%

K2O

%

Altitude Oil

%

Temper

ature

Plant

height 1 1.00 0.732

* 0.880

** 0.533 0.576 0.586 0.195 0.956

** 0.180 0.814

** 0.639

* 0.703

* 0.521 0.534 0.065 -0.181 -0.428 -0.297 0.593 0.568

2 1.00 0.685* 0.279 0.328 0.491 0.195 0.801

** 0.515 0.829

** 0.758

* 0.935

** 0.508 -0.050 0.235 -0.248 -0.105 -0.500 0.268 0.703*

3 1.00 0.413 0.604 0.507 0.342 0.789**

0.367 0.742* 0.672

* 0.708

* 0.454 0.513 0.016 -0.074 -0.248 -0.092 0.589 0.441

4 1.00 -0.011 0.829**

-0.314 0.436 -0.202 0.527 0.128 0.303 0.048 0.241 0.016 -0.178 -0.868**

-0.383 0.756* 0.364

5 1.00 0.345 0.856**

0.423 0.045 0.329 0.587 0.322 0.416 0.0313 -0.260 -0.045 0.158 -0.247 -0.087 0.558 6 1.00 0.174 0.450 0.024 0.663

* 0.424 0.461 0.115 -0.063 -0.105 -0.311 -0.582 -0.574 0.520 0.648*

7 1.00 0.050 0.309 0.088 0.466 0.117 0.168 -0.095 -0.237 -0.019 0.491 -0.138 -0.349 0.360

8 1.00 0.206 0.803**

0.654* 0.761

* 0.492 0.470 0.168 -0.107 -0.348 -0.265 0.519 0.539

9 1.00 0.316 0.289 0.311 0.038 -0.287 0.264 -0.168 0.302 0.116 0.172 -0.081

10 1.00 0.772**

0.778**

0.389 0.187 0.372 -0.215 -0.496 -0.411 0.603 0.657*

11 1.00 0.716* 0.212 0.117 0.349 0.233 0.005 -0.261 0.143 0.707*

12 1.00 0.614 0.041 0.064 -0.260 -0.120 -0.491 0.240 0.734*

13 1.00 0.270 -0.146 -0.626 -0.122 -0.562 0.004 0.537 14 1.00 0.097 0.260 -0.392 0.293 0.482 -0.073

15 1.00 0.368 -0.277 -0.006 0.236 0.041

16 1.00 0.210 0.547 -0.097 -0.239

17 1.00 0.321 -0.774**

-0.217

18 1.00 0.108 -0.835**

19 1.00 -0.033

20 1.00

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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141

Table 4.13 Simple correlation matrix of physical properties and micronutrients

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

pH EC OC% CEC WHC Sand Silt Clay Zn

total

Zn

DTPA

Zn

Plant

Fe

total

Fe

DTPA

Fe

Plant

Cu

total

Cu

DTPA

Cu

Plant

Mn

total

Mn

DTPA

Mn

Plant 1 1.00 0.398 -0.422 0.311 0.094 0.315 -0.061 -0.596 0.638* 0.623 0.564 0.195 0.622 0.383 0.465 0.552 0.271 0.535 0.460 0.541

2 1.00 0.406 0.137 0.305 -0.428 0.360 0.254 0.627 0.367 0.439 -0.072 0.538 -0.013 0.235 0.672* -0.063 0.506 0.629 0.314

3 1.00 -0.151 0.299 -0.428 0.445 0.358 -0.188 0.275 -0.194 -0.698* 0.062 -0.696* 0.042 -0.067 -0.196 -0.334 -0.030 -0.178

4 1.00 0.036 0.435 -0.224 -0.540 0.753* 0.583 0.764* 0.533 0.207 0.470 -0.115 0.720* 0.182 0.595 0.248 0.697*

5 1.00 -0.333 0.552 0.091 0.249 0.188 0.490 -0.346 0.557 -0.117 0.664* 0.215 0.504 0.043 0.463 0.100

6 1.00 -0.795** -0.825** 0.099 0.417 0.122 0.254 -0.239 0.140 -0.288 0.163 0.127 0.066 -0.125 0.518

7 1.00 0.374 -0.01 -0.439 0.101 -0.392 0.450 -0.297 09492 -0.148 0.045 -0.209 -0.029 -0.499

8 1.00 -0.266 -0.403 -0.391 -0.133 -0.189 -0.154 -0.118 -0.206 -0.220 -0.131 0.031 -0.524

9 1.00 0.732* 0.880** 0.533 0.576 0.586 0.195 0.956** 0.180 0.814** 0.639* 0.703*

10 1.00 0.685* 0.279 0.328 0.491 0.195 0.801** 0.515 0.829** 0.758* 0.935**

11 1.00 0.413 0.604 0.507 0.342 0.789** 0.367 0.742* 0.672* 0.708*

12 1.00 -0.011 0.829** -0.314 0.436 -0.202 0.527 0.128 0.303

13 1.00 0.345 0.856** 0.423 0.045 0.329 0.587 0.322

14 1.00 0.174 0.450 0.024 0.663* 0.424 0.461

15 1.00 0.050 0.309 0.088 0.466 0.117

16 1.00 0.206 0.803** 0.654* 0.761*

17 1.00 0.316 0.289 0.311

18 1.00 0.772** 0.778**

19 1.00 0.716*

20 1.00

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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142

Table 4.14 Simple correlation matrix of physical properties, macronutrients and microclimatic conditions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

N (av)

N(total)

%

P2O5 % K2O % Altitude Oil % Tempera

ture

Plant

height

pH EC OC% CEC Moisture

content

Sand Silt Clay

1 1.00 0.270 -0.146 -0.626 -0.122 -0.562 0.004 0.537 0.759* 0.322 -0.050 0.546 -0.158 0.503 -0.170 -0.774

**

2 1.00 0.097 0.260 -0.392 0.293 0.482 -0.073 0.206 0.584 0.210 0.378 0.210 -0.197 0.424 -0.027

3 1.00 0.368 -0.277 -0.006 0.236 0.041 0.166 0.363 -0.189 -0.297 -.038 -0.163 -0.082 0.314

4 1.00 0.210 0.547 -0.097 -0.239 -0.415 0.230 0.317 -0.439 0.461 -0.427 0.231 0.590

5 1.00 0.321 -0.774* -0.217 -0.197 -0.091 0.576 -0.376 0.555 -0.090 0.243 0.016

6 1.00 0.108 -0.835**

-0.671* -0.017 0.569 -0.141 0.480 -0.496 0.580 0.469

7 1.00 -0.033 0.097 0.119 -0.424 0.636* -0.049 -0.001 -0.010 -0.012

8 1.00 0.673 0.361 -0.338 0.284 -0.094 0.277 -0.406 -0.328

9 1.00 0.398 -0.422 0.311 0.094 0.315 -0.061 -0.596

10 1.00 0.406 0.137 0.305 -0.428 0.360 0.254

11 1.00 -0.151 0.299 -0.428 0.445 0.358

12 1.00 0.036 0.435 -0.224 -0.540

13 1.00 -0.333 0.552 0.091

14 1.00 -0.795**

-0.825**

15 1.00 0.374

16 1.00 * Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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4.5. Conclusions

The essential oil composition of aerial parts of ten samples of Origanum vulgare

L. family Lamiaceae, collected from different locations in Central Himalayas, India was

analyzed by GC and GC/MS. Cluster analysis was done to classify plants collected from

different locations on the basis of their principal components. Macro and micronutrients

(N, P, K, Zn, Cu, Fe and Mn) in soil and plant samples were also determined. Statistical

analysis of correlation coefficient was done to correlate different environmental and soil

factors with major constituents. The results of the present investigation are summarized

in this section.

Chemosystematics

Cluster analysis revealed variation in the essential oil composition of wild

Origanum vulgare L. collected from ten sites in Central Himalaya, India. The wild

Origanum is classified into four chemotypes as follows:

Chemotype I: Kilbury, Mukteshwar, Rushi, Mussoorie and Nainital

(Bicyclogermacrene, elemol, α-Cadinol, linalool, germacrene D,

bornyl acetate and (E)-caryophyllene)

Chemotype II: Ramgarh (Thymol, germacrene D, carvacrol, α-Cadinol, (E)-

caryophyllene and linalool)

Chemotype III: Dhoulchina and Champawat (p-Cymene, -terpinene, thymol

and carvacrol)

Chemotype IV: Dharchula and Munsiyari (Caryophyllene oxide, aliphatic

hydrocarbons and thymol)

Correlation among major constituents

p-Cymene is positively correlated with γ-terpinene and carvacrol while γ-

terpinene was found to be positively correlated with carvacrol. The correlation

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144

suggested that γ-terpinene and p-cymene are the biosynthetic precursors (via enzymatic

hydroxylation) of carvacrol.

Effect of macronutrient and micronutrients on essential oil composition

Correlation analysis suggested that the essential oil composition was affected by

variation in soil macro and micronutrients in soil and plant. Available nitrogen was

positively correlated with linalool, bornyl acetate, (E)-caryophyllene, germacrene D and

β-bisabolene while negatively correlated with thymol suggesting role of nitrogen in

their biosynthesis. Available potassium was found to be positively correlated with

thymol and caryophyllene oxide

Available Zn was found to be positively correlated with β-bisabolene. Total iron

(Fe) present in soil was positively correlated with p-cymene and ץ-terpinene. Total Mn

in plant was positively correlated with (E)-caryophyllene and β-bisabolene.

Effect of plant characteristics and microclimatic conditions on essential oil

composition

Percentage oil yield was negatively correlated with plant height, linalool and

(E)-caryophyllene while positively correlated with thymol, caryophyllene oxide and

aliphatic hydrocarbons. Plant height was positively correlated with (E)-caryophyllene

and germacrene D while negatively correlated with aliphatic hydrocarbons. Soil pH was

found to be negatively correlated with thymol and positively correlated with elemole.

Percent organic carbon was negatively correlated with p-cymene, (E)-ocimene and

caryophyllene oxide.

Thus, from our results it can be concluded that essential oil composition of

aromatic and medicinal plants are affected by variation in soil properties and

microclimatic conditions. Nitrogen, zinc and manganese in soil positively affect

biosynthesis of β-bisabolene while potassium increases the amount of thymol and

caryophyllene oxide and iron p-cymene and ץ-terpinene in the collected plant material.

Longer is the plant height, more will be the synthesis of (E)-caryophyllene and

germacrene D. In acidic soils, there will be more chances of synthesis of elemole as

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145

compared to other essential oil constituents. In organically poor soil, the plant

synthesizes more p-cymene, (E)-ocimene and caryophyllene oxide.

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