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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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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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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
Estelar
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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