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Chapter- 5

Effect of soil properties and microclimatic

conditions on essential oil composition of

Craniotome furcata (Link) O. Kuntze and its

chemosystematics

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5.1. Introduction

Craniotome furcata (Link.) O. Kuntze (Syn. C. versicolar, Anisomeles (Link)

furcata, A. nepalensis, Nepeta versicolor) belonging to family the Lamiaceae is an

erect, perennial, branched and soft hairy herb. It has 1-2 m tall stem with subwoody

base. Petiole is 2.5-7 cm long and leaves are stalked, cordate, broadly ovatewith dentate

margin. The flowers are numerous, white pink or yellow, crowed in small stalked cyme

forming narrow terminal panicles (14-18 cm). The lower floral leaves are leaf like while

spatulated upper leaves. The calyx (1.5 mm) is ovoid, glandular and 5-toothed. The

bracteoles are shorter than calyx tube. The corolla (3-4 mm) is reddish or purple-red in

colour. It is longer than the calyx, limb 2-lipped, upper lip very short, erect, hood like,

longer, spreading, 3-lobed and mid-lobe largest. The stamens are four, in unequal pairs,

ascending under the upper lip, outer or interior pair longer than the inner1,2

. Only one

species of C. furcata has been reported in India3,4

. It is distributed in China, Bhutan,

India, Korea, Laos, Myanmar, Nepal, Sikkim, Taiwan and Vietnam at a height of 1500-

2300 m 1,5

.

C. furcata has been used as folk medicine. The leaf juice is applied for

treatment of wounds 6. Joshi (2010)

7and Joshi et al. (2010)

8 reported the antimicrobial

and antioxidant activity in the essential oil and extract of C. furcata respectively.

Joshi and Pande (2009)9

reported the essential oil composition of C. furcata for

the first time from India. They reported germacren D (30.9%) as the major component

along with germacrene D-4-ol (12.1%), α-cadinol (6.4%), 3-octanone (6.1%),

germacrene A (5.8%) and epi-α-cadinol (4.0%). Three saponins, craniosaponin A and

buddlejasaponins Ia and I were isolated from the n-butanol soluble fraction of C.

furcata for the first time. Among them, craniosaponin A was identified as a new

compound10

. Cranioside A and B, mussaeniside and ningpogenin were isolated from the

ethyl acetate fraction of C. furcata and among them, cranioside A and B were identified

as new compounds11

.

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The essential oil composition of medicinal and aromatic plants is not constant

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

environmental factors like nature of soil, climatic conditions such as light, altitude,

moisture, growing and harvesting time etc. 12

. Important factors, which affects the

essential oil composition of aromatic and medicinal plants play a very important role in

the biogenetic pathways of different secondary metabolites of oil. Therefore, it is

necessary to investigate the relationship among the metal content in soil, plant and

environmental factors with the active constituents of aromatic and medicinal plants to

examine which factor is responsible for any variation of active constituents of aromatic

and medicinal plant.

Literature search revealed that no work has been done on the chemosystematics

of the plant Craniotome furcata (Link.) O. Kuntze with respect to soil and

microclimatic conditions in Uttarakhand region. Therefore, the objective of the present

work is to examine the effect of macro and micronutrient in soil and plant and

microclimatic conditions on essential oil composition of C. furcata,

5.2. Collection of plant material and soil samples

Fresh plant material of C. furcata (Link.) O. Kuntze along with its soil samples

(0-20 cm) were collected in September to November, 2010 from ten locations viz.

Bhowali (29º23'N: 79º31’E), Ramgarh (29º23'N: 79º30'E), Rushi village (29º23'N:

79º30'E), Nainital (29º23'N: 79º30'E), Jeolikot (29º23'N: 79º30'E), Mussoorie (30º 27'

N: 78º 06' E), Mukteshwar (29°28'N: 79°39'E), Kilbury (29º23'N: 79º30'E), Binsar

(29°37'N: 79º40'E) and Munsiyari (30°04'37"N: 80°23'04"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 in Botanical Survey of India, Dehradun (Voucher no. 34806).

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5.3. Fractionation of the oil and identification of major

compounds

The essential oils of C. furcata (5.0 mL) were fractionated by column

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

hexane (Scheme 5.1 and 5.2). The fractions (CF # 1 and CF# 2) obtained by column

chromatography were analyzed by spectroscopy (1H and

13C NMR) and MS to

determine their identity.

5.3.1. Flow sheet (1) for CC of essential oil C. furcata

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 (13-20) Fr (21-26) Fr (28-33) Fr (34-43)

A B C D E

CF # 01

Scheme 5.1 Isolation of compound from C. furcata (Link.) from Kilbury.

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5.3.2. Flow sheet (2) for CC of essential oil C. furcata (Link.).

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 (13-20) Fr (21-26) Fr (27-33) Fr (34-43)

A B C D E

Recolumn 10% Et2O

CF # 02

Scheme 5.2 Isolation of compound from C. furcata (Link.) from Rushi.

5.4. Results and Discussion

5.4.1. Characterization of the constituents

1) Characterization of CF#01:

Physico-chemical data

IR vmax cm-1

: 2927, 2871, 1689,1463, 1383, 980, 734 (Figure 5.1).

EIMS (70eV): 240(M+),189, 161 (100%), 133, 119, 105, 91, 79, 77, 67, 65, 43.

1H NMR (300MHz, CDCl3-TMS) (Figure 5.2):

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δ 0.89 (6H, d), 1.52 (3H, s), 4.80 (2H, dd), 4.80 (2H, dd), 5.24 (2H, m), 5.81 (1H, s).

13CNMR (75MHz, CDCl3-TMS) (Figure 5.3):

δ 129.8 (d, C-1), 29.7 (t, C-2), 34.5 (t, C-3), 148.8 (s, C-4), 135.6 (d, C-5), 133.2 (d, C-

6), 52.9 (d, C-7), 26.5 (t, C-8), 40.7 (t, C-9), 133.7 (s, C-10), 32.7 (d, C-11), 19.7 (q, C-

12), 20.9 (q, C-13), 15.8 (q, C-14), 109.0 (t, C-15).

The compound CF#01 was obtained as viscous liquid. The EIMS of compounds

shows molecular ion peak at m/z 204, corresponding to molecular formula C15H24. The

1H- NMR spectrum showed a doublet at δ 0.89 (6H, d) which revealed the presence of

isopropyl group in the molecule. The signal at δ 1.52 (3H, s) was attributed to a methyl

group attached to an olefinic carbon. The 13

C spectra of compounds represent fifteen

carbons. Based on these spectral data, the compound CF#01 was identified as

germacrene D. Finally, its identity was confirmed by comparison of its spectral data

with those reported in literature13, 14

.

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Figure 5.1 IR Spectrum of CF #1

Figure 5.2 1H NMR Spectrum of CF #1

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Figure 5.3 13

C NMR Spectrum of CF#1

2) Characterization of CF#02:

Physico-chemical data

IR vmax cm-1

: 3533, 2932, 1385, 1366, 1199, 980, 787, 764 (Figure 5.4).

EIMS (70eV): 222(M+), 204, 189, 161 (100%), 147, 133 123, 119, 105, 91, 81, 79, 77,

67, 43.

1H NMR (300MHz, CDCl3-TMS) (Figure 5.5):

δ 0.799 (d, 3H, Me-13), 0.819 (d, 3H, Me-12), 1.195 (s, 3H, Me-15), 1.542 (s, 3H, Me-

14), 4.933 (brd, 1H, H-1), 5.183 (dd, 1H, H-6), 5.233 (d, 1H, H-5).

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13CNMR (75MHz, CDCl3-TMS) (Figure 5.6):

δ 128.8 (d, C-1), 23.6 (t, C-2), 41.2 (t, C-3), 73.1 (s,C-4), 140.0 (d, C-5), 125.7 (d, C-6),

52.8 (d, C-7), 39.6 (t, C-8), 25.9 (t, C-9), 132.5 (s, C-10), 33.0 (d, C-11), 20.6 (q, C-12),

18.9 (q, C-13), 16.7 (q, C-14), 30.7 (q, C-15).

The compound CF#02 was obtained as light green liquid. The IR spectrum of

compound has a broad absorption band at 3533 cm-1

showing the presence of OH

group. The EIMS of the compound displayed (M+) at m/z 222 corresponding to the

molecular formula C15H26O. The 1H –NMR showed doublets for the signals at δ 0.819

(3H, d) and 0.799 (3H,d) for two methyl of isopropyl group. A signal at δ 1.195 (3H, s)

was attributed to a methyl attached with quaternary carbon bearing alcoholic group.

Another broad singlet at δ 1.542 (3H, s) appears for methyl attached to the carbon

having endocyclic double bond, which is confirmed by the broad doublet at δ 4.933

(1H, brd, H-1). One doublet appear at δ 5.233 (1H, d) and one doublet signal at δ 5.183

(1H, dd), clearly indicate the presence of one other endocyclic double bond. 13

C NMR

showed the presence of total fifteen carbons. On the basis of the above spectral data

CF#02 was characterized as germacrene D-4-ol. Finally, its identity was confirmed by

comparison of its spectral data with those reported in literature 15, 16.

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Figure 5.4 IR Spectrum of CF #2

Figure 5.5 1H NMR Spectrum of CF #2

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Figure 5.6 13

C NMR Spectrum of CF#2

5.4.2. Chemosystematics of Craniotome furcata.

The essential oils of C. furcata (Link.) O. Kuntze collected from ten sites were

analyzed by GC and GC/MS. The structures of major components are shown in

Figure 5.7. Ward’s hierarchical clustering analysis of major constituents of essential

oils was conducted in order to classify chemotypes (Figure 5.8). The result of cluster

analysis showed four groups on the basis of difference in their main chemical

constituents and allowing them to be characterized into four distinct chemotypes

(Table 5.1). Group one consisted oils of C. furcata collected from Binsar (Figure

5.9) and Munsiyari (Figure 5.10) (Chemotype I) was significantly rich in δ-elemene

(9.9-11.1%) and germacrene D (52.8-59.8%) while the second group consisted

Jeolikot (Figure 5.11), Mussoorie (Figure 5.12), Mukteshwar (Figure 5.13) and

Kilbury (Figure 5.14) (Chemotype II) which was further divided in to two subgroups

on the basis of their similarity in dendrogram. The main components of the first

subgroup (Jeolikot and Mussoorie) were δ-elemene (3.4-7.9%), germacrene D (36.7-

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36.8%), α-zinziberene (3.5-12.8%) and α-cadinol (1.1-9.3%) while the components of

second subgroup (Mukteshwar and Kilbury) was germacrene D (42.9-46.2%), α-

zinziberene (5.5-5.8%), germacrene B (1.6-11.9%) and α-muurolol (8.5-10.5%), so

from above discussion it is possible to integrate these in to two subgroups. The oil

from Nainital (Figure 5.15) and Rushi village (Figure 5.16) (Chemotype III) showed

the presence of γ-cadinene (6.6-9.6%), germacrene D-4-ol (10.0-24.8%), α-muurolol

(2.4-5.2%), α-cadinol (9.2-11.9%), oplopanon (5.2-6.2%) and α-bisabolol oxide A

(6.1-10.6%) were placed into group III. The plants having high content of δ-elemene

(3.0-6.8), germacrene D (13.3-17.5%), α-zinziberene (5.1-14.0%), germacrene B (3.5-

15.6%), α-muurolol (8.1-15.2%) and α-cadinol (2.3-8.6 %) from Bhowali (Figure

5.17) and Ramgarh (Figure 5.18) (Chemotype IV) belongs to group fourth.

The cluster analysis classified the essential oils into four chemotypes on the

basis of presence or absence of chemical markers.

Chemotype-I: δ-elemene and germacrene D

Chemotype-II: Subgroup-I: δ-elemene, germacrene D, α-zinziberene and α-cadinol

Subgroup-II: germacrene D, α-zinziberene, germacrene B and α-muurolol

Chemotype-III: γ-cadinene, germacrene D-4-ol, α-muurolol, α-cadinol, oplopanon and

α-bisabolol oxide A

Chemotype-IV: δ-elemene, germacrene D, α-zinziberene, germacrene B, α-muurolol

and α-cadinol

These four chemotypes showed the chemical variability in essential oil

composition of C. furcata collected from ten regions. Earlier reports showed the

presence of germacrene D, germacrene D-4-ol, epi-α-cadinol, and α-cadinol as major

constituents from Nainital9,17

.

It has been well documented that germacrene D plays an important role as a

precursor of various sesquiterpenes such as cadinenes and selinenes18,19

. Plant

terpenes have been found to show anti-herbivore defenses20

. Germacrene D has also

been reported to have deterrent effects against herbivores and insecticidal activity

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against mosquitoes21

, as well as repellent activity against aphids22

and ticks23

.

Therefore, the common presence of germacrene D in Craniotome furcata may

be useful as a source of other terpenes that could make the plants inedible for

herbivores24

.

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H

H

δ-elemene (5) α-cubebene (6) germacrene D (16)

MF- C15H24 MF- C15H24 MF- C15H24

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

(Z)

H

H

α-Zinziberene (17) (Z)-α-Bisabolene (23) ץ-cadinene (26)

MF- C15H24 MF- C15H24 MF- C15H24

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

OH HO

Germacrene B (30) Germacrene D-4ol (31) α-Muurolol (34)

MF- C15H24 MF- C15H26O MF- C15H26O

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

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H

H

OH

O

HO

H

α-Cadinol (35) α-Bisabolol oxide A (41)

MF- C15H26O MF- C15H26O2

FW- 222 g/mol FW- 238 g/mol

Figure 5.7 Structures of major constituents

Figure 5.8 Agglomerative hierarchical clustering analysis by SPSS 16.0 for the

chemical abundances of 12 essential oil components in the 10

populations of C. furcata.

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S.No

.

Compoundsa

RIb

RIc

Chemotype I Chemotype II Chemotype III Chemotype IV

Binsar

(9)

Munsiy

ari (10)

Jeoliko

t (1)

Musso

orie (5)

Muktesh

war (4)

Kilbury

(8)

Nainita

l (6)

Rushi

Village

(7)

Bhow

ali

(2)

Ramgar

h (3)

1 1-octene-3-ol AA 979 974 0.4 0.2 0.8 3.2 1.7 3.1 0.7 0.2 1.4 0.3

2 sabinene

MH 975 946 - - - - - 0.4 - - - -

3 (Z)- β-

ocimene

MH 1037 1032 - - 1.4 1.3 1.7 - - - - 0.3

4 (E)- β-

ocimene

MH 1050 1044 0.9 0.9 - - - - 1.6 0.4 - -

5 δ-elemene SH 1338 1335 11.1 9.9 7.9 3.4 2.7 3.3 2.2 1.7 3.0 6.8

6 α-cubebene SH 1348 1345 1.9 0.9 1.1 1.3 3.2 1.5 3.3 0.5 3.3 5.4

7 α-copaene SH 1376 1374 2.0 1.7 - - - - - - 1.7 -

8 β-bourbonene SH 1388 1387 3.5 4.5 - - - - - - - -

9 β–elemene SH 1389 1388 4.2 3.2 1.4 1.6 0.5 - 3.8 2.5 0.5 1.1

10 (E)-

caryophyllene

SH 1419 1417 3.3 2.7 1.03 0.7 1.2 1.0 4.2 1.6 1.9 0.5

11 γ-elemene SH 1436 1434 1.9 2.1 - 0.3 - - - - - -

12 α-guainene SH 1439 1437 - 0.8 - 0.7 0.7 - - - - 1.3

13 (Z)-β-

farnesene

SH 1442 1440 - - 0.9 - - - - - - -

14 9-epi-(E)-

caryophyllene

SH 1466 1464 - - - 1.2 - - - - - -

15 ar-curcumene SH 1480 1479 - - 1.7 3.5 - - - - - -

Table 5.1 Chemotypes of C. furcata collected from different sites

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16 germacrene D SH 1485 1484 52.8 59.8 36.7 36.8 46.2 42.9 4.0 4.5 13.3 17.5

17 α-zinziberene SH 1493 1493 - - 12.8 3.5 5.5 5.8 3.7 1.8 5.1 14.0

18 epi-cubibol OS 1494 1493 1.7 - - - - - - - - -

19 bicyclogerma

crene

SH 1500 1500 - - - - - 0.9 - - - -

20 α-muurolene SH 1500 1500 - - - 0.8 - 3.3 0.7 0.7 - -

21 (E)-β-guaiene SH 1502 1502 - - - - - 1.4 - - - -

22 α-fernesene SH 1505 1505 - - 1.3 0.6 - 1.1 2.8 1.0 - -

23 (Z)-α-

bisabolene

SH 1507 1506 - - - - - 10.3 - - - -

24 germacrene A SH 1509 1508 2.8 0.3 - - - - - - 2.78 -

25 δ-amorphene SH 1512 1511 - - 2.1 3.8 - - - - - -

26 γ-cadinene SH 1513 1513 - - 0.9 0.9 - 6.8 6.6 9.6 8.8 -

27 β-

sesquiphellan

drene

SH 1522 1521 - - 3.0 2.3 1.0 1.1 2.6 0.9 4.5 3.1

28 δ-cadinene SH 1523 1522 1.4 0.6 1.0 1.3 - - - - 2.5 -

29 hedycaryol OS 1548 1546 - - - 0.3 - - - - - -

30 germacrene B SH 1561 1559 1.5 1.7 1.0 4.1 11.9 1.6 4.8 6.9 3.5 15.6

31 germacrene

D-4-ol

OS 1575 1574 - - - 4.4 1.5 10.0 24.8 2.9 2.2

32 1,10-di-epi-

cubinol

OS 1619 1618 - - - - - 0.8 - - - -

33 α-muurolol OS 1646 1644 - - 2.7 3.8 8.5 10.5 5.2 2.4 15.2 8.1

34 β-eudesmol OS 1650 1649 - - 2.6 - - - - - - -

35 α-cadinol OS 1654 1652 0.7 - 1.1 9.3 1.5 0.9 9.2 12.0 8.6 2.3

36 ar-tumerone OS 1669 1668 - - - 0.6 3.2 - 4.6 1.3 3.3 1.9

37 epi-β-

bisabolol

OS 1671 1670 - - - 1.6 1.2 - 3.7 1.9 3.6 2.2

38 khusinol OS 1680 1679 - - 1.2 - - - - - -

39 (Z)-(E)-α- OS 1690 1690 1.1 0.6 - - - - - - - -

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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, %), AA=

aliphatic alcohol, MH= monoterpene hydrocarbon, OM= oxygenated monoterpene, SH= sesquiterpene hydrocarbon, OS= oxygenated sesquiterpene.

bergamotol

40 oplopanone OS 1740 1739 - - - 0.7 2.7 - 5.2 6.2 1.7 3.3

41 α-bisabolol

oxide A

OS 1749 1748 - - - - - - 6.1 10.6 - -

Total 91.2 89.9 81.4 93.2 94.9 96.7 81.4 93.2 94.9 96.7

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Figure 5.9 GC of the essential oil of C. furcata collected from Binsar.

Figure 5.10 GC of the essential oil of C. furcata collected from Munsiyari.

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Figure 5.11 GC of the essential oil of C. furcata collected from Jeolikot.

Figure 5.12 GC of the essential oil of C. furcata collected from Mussoorie.

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Figure 5.13 GC of the essential oil of C. furcata collected from Mukteshwar.

Figure 5.14 GC of the essential oil of C. furcata collected from Kilbury.

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Figure 5.15 GC of the essential oil of C. furcata collected from Nainital.

Figure 5.16 GC of the essential oil of C. furcata collected from Rushi

village.

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Figure 5.17 GC of the essential oil of C. furcata collected from Bhowali.

Figure 5.18 GC of the essential oil of C. furcata collected from Ramgarh.

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5.4.3. Physicochemical properties of soil

Physicochemical properties of the soil are given in Table 5.2. Soils were

classified as loamy sand and sandy loam. Soils were acidic to neutral (pH 5.42 to 7.85).

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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Table 5.2. Physicochemical properties of soil used in the study

Sites

Binsar Munsiyari Jeolikot Mussoorie Mukteshwar Kilbury Nainital

Rushi

Bhowali

Ramgarh

General soil properties

Sand (%) 66 70 78 68 82 84 78 76 80 70

Silt (%) 20 26 15 26 16 14 12 18 15 22

Clay (%) 16 8 4 6 4 2 8 6 5 8

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

Other soil properties

pH (1:2) 5.83±0.27 5.42±0.010 7.85±0.02 7.62±0.33 6.1±0.80 6.83±0.06 6.84±0.69 7.44±0.04 6.32±0.03 6.41±0.07

O.C. % 1.71±0.10 4.17±0.04 1.68±0.03 3.04±0.14 3.85±0.04 1.2±0.00 3.23±0.20 2.65±0.35 2.69±0.06 3.15±0.04

EC 0.111±0.000 0.11±0.001 0.42±0.07 0.156±0.011 0.054±0.022 0.23±0.040 0.074±0.001 0.34±0.02 0.78±0.01 0.195±0.01

CEC 10.26±0.030 27.28±0.070 10.78±0.010 18.13±0.060 16.3±0.100 31.97±0.040 25.76±0.080 38.11±0.010 13.64±0.100 15.06±0.130

W HC 37.92±0.070 40.36±0.010 43.52±0.030 58.51±0.100 38.24±0.080 49.69±0.010 39.57±0.050 46.08±0.010 42.88±0.070 35.11±0.040

Total content (mg kg-1)

Zn 57.663±0.028 38.67±0.002 25.05±0.16 54.281±0.05 26.875±0.10 62.657±0.03 47.377±0.26 91.677±0.01 42.007±0.11 41.718±0.05

Fe 561.849±0.228 519.33±0.230

521.664±0.01 522.604±0.01 516.859±0.26 566.618±0.04 542.206±0.03

559.248±0.05 556.288±0.03 528.632±0.06

Mn 15.5±0.130 24.63±0.090 10.70±0.10 19.825±0.29 10.7±0.010 25.783±0.01 14.67±0.06 15.55±0.08 11.484±0.05 10.825±0.01

Cu 158.336±0.02 165±0.73 192.105±0.08 220.897±0.09 182.15±0.02 275.888±0.07 303.703±0.09 348.023±0.03 255.251±0.12 221.418±0.05

Available content (mg kg-

1)

Zn 2.710±0.05 0.784±0.03 3.954±0.03 0.984±0.05 1.706±0.01 9.452±0.01 7.3680.03 12.262±0.05 1.486±0.03 1.104±0.01

Fe 22.65±0.04 36.68±0.07 32.53±0.04 91.70±1.03 29.00±0.08 29.94±0.11 28.77±0.06 57.98±0.30 35.38±0.40 33.28±0.08

Mn 7.08±0.06 14.28±0.11 6.68±0.05 17.11±0.88 3.00±0.73 14.56±0.21 15.43±0.42 17.39±0.20 9.58±0.07 10.00±0.23

Cu 0.150±0.03 1.119±0.01 0.96±0.02 1.820±0.03 0.320±0.04 2.256±0.11 1.830±0.02 7.328±0.08 0.610±0.07 0.400±0.00

Macronutrient content (%)

N (av) 0.005±0.10 0.008±0.03

0.009±0.05 0.012±0.34 0.012±0.71 0.011±0.69 0.010±0.63 0.014±0.01 0.009±0.25 0.012±0.09

N(tot) 0.20±0.01 0.28±0.01 0.20±0.04 0.25±0.03 0.24±0.01 0.21±0.04 0.18±0.05 0.35±0.03 0.13±0.02 0.30±0.06

P (av) 0.0026±0.00 0.0011±0.00 0.0014±0.00 0.0009±0.00 0.0007±0.00 0.0037±0.00 0.0019±0.001 0.0024±0.01 0.0006±0.00 0.0033±0.00

K (av) 0.0163±0.00 0.016±0.001 0.0076±0.00 0.0176±0.004 0.0046±0.001 0.0255±0.00 0.0132±0.00 0.009±0.002 0.0242±0.001 0.0193±0.001

*(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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177

5.4.4 Microclimatic conditions and oil properties

Microclimatic conditions and oil properties are shown in Table 5.3.

Table 5.3 Microclimatic conditions and oil properties

Microclim

atic and

other

properties

Binsar Muns

iyari

Jeoli

kot

Musso

Orie

Mukte

shwar

Kilb

ury

Nai

nital

Rushi Bho

wali

Ramg

arh

Altitude

(m)

2300 2386 1490 2000 2265 2200 2100 1600 1706 1789

Temperatu

re (0C)

23 18 30 25 20 22 23 28 28 23

Plant

height

(inch)

54.67

±1.53

37.67

±2.52

46

±2.65

35.33

±3.51

34.33

±2.52

26.67

±1.53

27.33

±1.53

39.33

±3.51

26

±2.66

34

±2.00

Month of

collection

in 2010

Octo

ber

Octo

ber

Septe

mber

Octo

Ber

Novem

ber

Novem

ber

Novem

ber

Septem

ber

Novem

ber

Novem

ber

Oil colour yellow yellow yellow yellow yellow light

green

light

green

yellow yellow yellow

Oil % 0.43 0.45 0.30 0.52 0.38 0.36 0.34 0.35 0.32 0.32

5.4.5. Correlation among major constituents

Simple correlation matrix (r) among major constituents of different chemotypes

given in (Table 5.4). δ-elemene is positively correlated with germacrene D (r=0.641,

P≤0.05) and negatively correlated with γ-cadinene (r=-0.678, P≤0.05), oplopanon (r=-

0.648, P≤0.05) and α-cadinol (r=-0.724, P≤0.05). Germacrene D showed negative

correlation with γ-cadinene (r=-0.695, P≤0.05), germacrene D-4-ol (r=-0.682, P≤0.05),

α-cadinol (r=-0.784, P≤0.01), oplopanon (r=-0.733, P≤0.05) and α-Bisabolol oxide A

(r=-0.678, P≤0.05). γ-cadinene is positively correlated with Germacrene D-4-ol

(r=0.635, P≤0.05), α-cadinol (r=0.675, P≤0.05) and α-Bisabolol oxide A (r=0.640,

P≤0.05). Germacrene D-4-ol is positively correlated with α-cadinol (r=0.780, P≤0.01),

oplopanon (r=0.866, P≤0.01) and α-Bisabolol oxide A (r=0.966, P≤0.01) while α-

cadinol is positively correlated with oplopanon (r=0.694, P≤0.05) and α-Bisabolol oxide

A (r=0.702, P≤0.05). Oplopanon showed positive correlation with α-Bisabolol oxide A

(r=0.906, P≤0.01).

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178

5.4.6. Effect of macronutrient on essential oil composition

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

are given in Table 5.5 Available N is negatively correlated with δ-elemene (r=-0.734,

P=<0.05).

5.4.7. Effect of micronutrient on essential oil composition

5.4.7.1. Effect of zinc (Zn)

Total Zn in soil is positively correlated with Germacrene D-4-ol (r=0.735,

P≤0.05) and α-Bisabolol oxide A (r=0.695, P≤0.05) and available Zn is positively

correlated with γ-cadinene (r=0.722, P≤0.05), Germacrene D-4-ol (r=0.703, P≤0.05)

and α-Bisabolol oxide A (r=-0.778, P≤0.01) while total Zn in plants is positively

correlated with γ-cadinene (r=0.642, P≤0.05) (Table 5.6). 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 biosynthesis25

. As 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 synthesis26

.

5.4.7.2. Effect of iron (Fe)

Total iron in soil showed positive correlation with γ-cadinene (r=0.699, P≤0.05)

(Table 5.7).

5.4.7.3. Effect of copper (Cu)

. Available Cu and showed positive correlation with γ-cadinene (r=0.639,

P≤0.05), germacrene D-4-ol (r=0.912, P≤0.01), oplopanon (r=0.871, P≤0.01) and α-

bisabolol oxide A(r=0.705, P≤0.05) while Cu in plant is positively correlated with

Estelar

179

germacrene D-4-ol ( r=0.705, P≤0.01) and α-bisabolol oxide A (r=0.712, P≤0.05)

(Table 5.8).

5.4.7.4. Effect of manganese (Mn)

Total Mn in soil is negatively correlated with δ-elemene (r=-0.766, P=<0.01)

and germacrene D (r=-0.825, P=<0.01) while positively correlated with γ-cadinene

(r=0.886, P≤0.01), germacrene D-4-ol (r=-0.791, P=<0.01), α-cadinol (r=-0.752,

P=<0.05), oplopanon (r=0.748, P≤0.05) and α-bisabolol oxide A (r=-0.800, P=<0.01)

while Mn in plants is positively correlated with γ-cadinene (r=0.716, P≤0.05) (Table

5.9). Duarte et al. (2010)27

suggested that γ-cadinene, limonene, and caryophyllene

oxide have a strong relationship with micronutrient balance in soils (Zn, Cu, Fe, Mn) in

Eugenia dysenterica.

5.4.8. Effect of plant properties and microclimatic conditions on essential oil

composition

Altitude is positively correlated with germacrene D (r=0.644, P≤0.05)and

negatively with temperature (r=-0.909, P≤0.01) while plant height is positively

correlated with δ-elemene (r=0.723, P≤0.05) and negatively correlated with α-muurolol

(r=-0.759, P≤0.05) (Table 5.10).

Comparison of volatile constituents of C. furcata from ten locations shows that

there is some quantitative difference between the essential oil components which may

be due to the environmental factors. The only distinct feature is altitudinal variation

with Jeolikot being located at 1490 m, Rushi, 1600 m, Mussoorie, 2000 m, Nainital,

2100 m, Kilbury, 2220 m, Mukteshwar, 2265, Binsar, 2300 m and Munsiyari, 2386 m,

having germacrene D 13.3%, 17.5%, 36.7%, 36.8 %, 46.2%, 42.9%, 52.8% and 59.8 %

respectively. This clearly indicates a continuous increasein the percentage of

germacrene D with increasing altitude, Vakou et al. (1993)28

reported that altitude

influenced the oil content of O.vulgare ssp. hirtum from Grees..

5.4.9. Effect of soil physical properties on essential oil composition

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180

Soil CEC is positively correlated with germacrene D-4-ol (r=0.651, P≤0.05) and

α-bisabolol oxide A (r=0.683, P≤0.05). Sand is positively correlated with α-muurolol

(r=0.637, P≤0.05) (Table 5.11).

5.4.10. Micro, macro nutrients and microclimatic conditions

Total zinc in soil is positively correlated with available Zn in soil (r=0.728,

P≤0.05), Zn concentration in plant (r=0.937, P≤0.01), total iron in soil (r=0.695,

P≤0.05), available Cu in soil (r=0.831, P≤0.01), Cu in plant (r=0.781, P≤0.01) and total

Mn in soil (r=0.676, P≤0.05) (Table 5.12). Available Zn in soil is positively correlated

with Zn concentration in plant (r=0.767, P≤0.01), available Cu in soil (r=0.815,

P=<0.01), Cu in plant (r=0.738, P≤0.01), and total Mn in soil(r=0.810, P≤0.01). Zinc

concentration in plant is positively correlated with total iron in soil (r=0.741, P≤0.05),

available Cu in soil (r=0.755, P≤0.05), Cu in plant (r=0.727, P≤0.05) and total Mn in

soil(r=0.723, P≤0.05). Available Fe in soil is positively correlated with Fe in plant

(r=0.776, P≤0.01). Iron in plant is negatively correlated with altitude (r=-0.682,

P≤0.05). Available Cu in soil is positively correlated with Cu in plant (r=0.862,

P≤0.01), total Mn in soil (r=0.775, P≤0.01) and available Mn in soil (r=0.646, P≤0.05).

Copper in plant is positively correlated with total Mn in soil (r=0.694, P≤0.05),

available Mn in soil (r=0.808, P≤0.01) and Mn in plant (r=0.730, P≤0.05). Total Mn in

soil is positively correlated with available Mn in soil (r=0.633, P≤0.05) and Mn in plant

(r=0.662, P≤0.05). Available Mn in soil is positively correlated with Mn in plant

(r=0.750, P≤0.05). Altitude is negatively correlated with temperature (r=-0.909,

P≤0.01).

5.4.11. Physical properties and micronutrients

Percent organic carbon is negatively correlated with total iron in soil (r=-0.643,

P≤0.05) (Table 5.13). Cation exchange capacity is positively correlated with total Zn in

soil (r=0.688, P=<0.05), available Zn in soil (r=0.764, P≤0.05), Zn concentration in

plant (r=0.680, P≤0.05), available Cu in soil (r=0.805, P≤0.01), Cu in plant (r=0.977,

P≤0.05), total Mn in soil (r=0.696, P≤0.05) and Mn in plant (r=0.685, P≤0.05). Water

Estelar

181

holding capacity is positively correlated with available iron (r=0.812, P≤0.01). Sand is

negatively correlated with silt and clay percentage (r=-0.812, P≤0.01 and r=-0.770,

P≤0.01 respectively).

5.4.12. Physical properties, macronutrients and microclimatic conditions

Soil pH is negatively correlated with altitude (r=-0.697, P≤0.05) and

positively correlated with temperature (r=-0.742, P≤0.05) (Table 5.14). Silt is

positively correlated with oil percentage (r=-0.741, P≤0.05). Clay is positively

correlated with plant height (r=0.638, P≤0.05) while negatively correlated with

available nitrogen (r=-0.628, P≤0.05).

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182

Table-5.4 Simple correlation matrix (r) among major constituents.

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

S.N δ-elemene α-

cubeben

e

germacrene

D

α-zinziberene Z-α-

bisabolene

γ-cadinene germacrene

B

germacrene

D-4-ol

α-muurolol α-cadinol oplopanon α-bisabolol

oxide A

1 1.00 0.137 0.641* -0.018 -0.195 -0.678

* -0.275 -0.549 -0.564 -0.702

* -0.648

* -0.488

2 1.00 -0.184 0.335 -0.206 -0.240 0.581 -0.308 0.362 -0.177 -0.059 -0.289

3 1.00 -0.235 0.199 -0.695* -0.335 -0.682* -0.364 -0.789** -0.733

* -0.678*

4 1.00 0.043 -0.198 0.396 -0.264 0.360 -0.234 -0.223 -0.287

5 1.00 0.298 -0.255 -0.208 0.354 -0.288 -0.249 -0.160

6 1.00 -0.183 0.635* 0.441 0.675

* 0.627 0.640

*

7 1.00 0.143 0.276 0.041 0.292 0.046

8 1.00 -0.172 0.780**

0.866**

0.966**

9 1.00 0.163 0.032 -0.226

10 1.00 0.694* 0.702

*

11 1.00 0.906**

12 1.00 * Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

Table-5.5 Correlation matrix (r) between macronutrients and major constituents of essential oil

1

N

(av)

2

N(total)

%

3

P2O5

%(av)

4

K2O %

(av)

5

δ-

eleme

ne

6

α-

cubebe

ne

7

germacre

ne D

8

α-

zinzibere

ne

9

Z-α-

bisabolen

e

10

γ-

cadinene

11

germacre

ne B

12

germacre

ne D-4-ol

13

α-

muurolol

14

α-

cadinol

15

oplopan

on

16

α-

bisabolol

oxide A

1 1.00 0.588 0.093 -0.141 -0.732* -0.159 -0.479 0.277 0.112 0.307 0.602 0.552 0.308 0.477 0.456 0.416

2 1.00 0.278 -0.326 0.009 -0.247 -0.031 -0.026 -0.131 -0.149 0.416 0.518 -0.457 0.085 0.238 0.421

3 1.00 0.349 0.110 0.221 -0.111 0.208 0.586 0.139 0.041 0.095 -0.020 -0.234 -0.031 0.164

4 1.00 0.032 0.205 -0.014 -0.032 0.521 0.269 -0.314 -0.341 0.493 -0.44 -0.427 -0.353

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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183

Table-5.6 Correlation matrix (r) among zinc (Zn) in soil and plant with major constituents in soil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level

Table-5.7 Correlation matrix (r) among iron (Fe) in soil and plant with major constituents in soil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

1

Zn

Total

2

Zn

DTPA

3

Zn

Plant

4

δ-

elemene

5

α-

cubebene

6

germacrene

D

7

α-

zinziberene

8

Z-α-

bisabole

ne

9

γ-

cadine

ne

10

germacr

ene B

11

germacrene D-

4-ol

12

α-

muurolol

13

α-

cadinol

13

oplopano

n

14

α-

bisabolol

oxide A

1 1.00 0.728* 0.937** -0.306 -0.311 -0.355 -0.463 0.251 0.594 -0.175 0.735* -0.167 0.525 0.541 0.695*

2 1.00 0.767** -0.477 -0.411 -0.452 -0.157 0.454 0.722* -0.185 0.703* -0.044 0.380 0.387 0.778**

3 1.00 -0.458 -0.254 -0.349 -0.288 0.513 0.642* -0.096 0.611 0.091 0.449 0.461 0.561

1

Fe

total

2

Fe

DTPA

3

Fe

Plant

4

δ-

elemene

5

α-

cubebene

6

germacrene

D

7

α-

zinziberene

8

Z-α-

bisabolene

9

γ-

cadinene

10

germacrene

B

11

germacrene

D-4-ol

12

α-

muurolol

13

α-

cadinol

14

oplopanon

15

α-

bisabolol

oxide A

1 1.00 -0.209 -0.055 -0.175 0.036 -0.294 -0.324 0.479 0.699* -0.363 0.293 0.248 0.234 0.230 0.343

2 1.00 0.776** -0.339 -0.462 -0.135 -0.169 -0.169 0.048 -0.053 0.363 -0.163 0.587 0.033 0.185 3 1.00 -0.340 -0.233 -0.442 0.186 -0.269 0.242 0.134 0.443 0.135 0.613 0.113 0.232

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184

Table-5.8 Correlation matrix (r) among copper (Cu) in soil and plant with major constituents in soil

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

Table-5.9 Correlation matrix (r) among manganese (Mn) in soil and plant with major constituents in soil

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

1

Cu

total

2

Cu

DTPA

3

Cu

Plant

4

δ-

elemene

5

α-

cubebene

6

germacrene

D

7

α-

zinziberene

8

Z-α-

bisabolene

9

γ-

cadinene

10

germacrene

B

11

germacrene

D-4-ol

12

α-

muurolol

13

α-

cadinol

14

oplopanon

15

α-

bisabolol

oxide A

1 1.00 0.202 0.588 0.105 -0.537 0.449 -0.516 0.607 0.065 -0.517 -0.100 -0.228 -0.175 -0.292 -0.066

2 1.00 0.862** -0.483 -0.581 -0.480 -0.280 0.096 0.639* -0.073 0.912** -0.201 0.622 0.664* 0.871** 3 1.00 -0.383 -0.578 -0.277 -0.444 0.328 0.591 -0.164 0.705* -0.170 0.423 0.505 0.712*

1

Mn

total

2

Mn

DTPA

3

Mn

Plant

4

δ-

elemene

5

α-

cubebene

6

germacrene

D

7

α-

zinziberene

8

Z-α-

bisabolene

9

γ-

cadinene

10

germacrene

B

11

germacrene

D-4-ol

12

α-

muurolol

13

α-

cadinol

14

oplopanon

15

α-

bisabolol

oxide A

1 1.00 0.633* 0.662* -0.766** -0.203 -0.825** -0.042 0.243 0.886** 0.019 0.791** 0.296 0.754* 0.748* 0.800**

2 1.00 0.750* -0.336 -0.480 -0.348 -0.363 0.217 0.481 -0.334 0.533 -0.165 0.560 0.285 0.528

3 1.00 -0.280 -0.353 -0.505 -0.351 0.069 0.716* -0.374 0.521 0.042 0.513 0.468 0.592

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185

Table-5.10 Correlation matrix (r) between microclimatic conditions and major constituents of essential oil

1

Alti

tude

2

Oil

%

3

Temp

4

Plant

height

5

δ-

eleme

ne

6

α-

cubebene

7

germacre

ne D

8

α-

Zinziberene

9

Z-α-

bisabolene

10

γ-

cadinene

11

germacrene

B

12

germacrene

D-4-ol

13

α-

muurolol

14

α-

cadinol

15

oplopano

n

16

α-Bisabolol

oxide A

1 1.00 0.588 -0.909** 0.001 0.262 0.115 0.644* -0.574 0.239 -0.372 -0.039 -0.395 -0.203 -0.437 -0.243 -0.319

2 1.00 -0.437 0.233 0.212 -0.274 0.553 -0.615 -0.086 -0.418 -0.207 -0.137 -0.461 -0.004 -0.335 -0.223

3 1.00 0.131 -0.234 -0.197 -0.548 0.348 -0.186 0.434 -0.244 0.350 0.143 0.486 0.188 0.291 4 1.00 0.723* -0.068 0.440 -0.128 -0.369 -0.369 -0.211 -0.070 -0.759* -0.354 -0.262 -0.066

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

Table 5.11 Correlation matrix (r) between physical properties of soil and major constituents of essential oil

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

pH EC OC% CEC Moist

ure

conten

t

sand silt clay δ-

elem

ene

α-

cube

bene

germ

acren

e D

α-

Zinzi

beren

e

Z-α-

bisabo

lene

γ-

cadinen

e

germacr

ene B

germacre

ne D-4-

ol

α-

muurolol

α-

cadinol

oplopano

n

α-

Bisabolo

l oxide A

1 1.00 0.240 -0.411 0.131 0.621 0.200 -0.228 -0.457 -0.450 -0.432 -0.442 0.379 0.072 0.294 -0.147 0.406 -0.017 0.482 0.204 0.351

2 1.00 -0.312 -0.161 0.129 0.326 -0.298 -0.330 -0.200 -0.085 -0.402 0.237 -0.027 0.546 -0.254 0.056 0.557 0.307 0.006 -0.010 3 1.00 0.080 -0.254 -0.187 0.414 0.013 -0.102 0.104 -0.014 -0.193 -0.560 -0.260 0.500 0.109 -0.089 0.159 0.258 0.064

4 1.00 0.287 0.271 -0.064 -0.311 -0.452 -0.549 -0.254 -0.393 0.415 0.584 -0.096 0.651* -0.097 0.355 0.531 0.683*

5 1.00 0.026 0.205 -0.415 -0.389 -0.647 0.026 -0.184 0.333 0.237 -0.410 0.154 0.018 0.404 -0.142 0.038

6 1.00 -0.812** -0.770** -0.630 -0.160 -0.246 0.232 0.493 0.543 0.081 0.081 0.637* 0.070 0.322 0.123

7 1.00 0.341 0.461 -0.114 0.428 -0.220 -0.312 -0.565 0.038 -0.133 -0.474 -0.128 -0.438 -0.264

8 1.00 0.634 0.421 0.172 -0.400 -0.431 -0.344 -0.122 -0.054 -0.544 -0.137 -0.085 0.004

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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186

Table 5.12 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

N

(av)

N (t)

%

P2O5

%

K2O % Zn Zn

DTPA

Zn

Plant

Fe Fe

DTPA

Fe

Plant

Cu Cu

DTPA

Cu

Plant

Mn Mn

DTPA

Mn

Plant

Altitude Oil

%

Temper

ature

Plant

height 1 1.00 0.588 0.093 -0.141 0.307 0.341 0.425 -0.151 0.477 0.568 -0.055 0.556 0.473 0.584 0.355 0.141 -0.342 -0.133 0.092 -0.516

2 1.00 0.278 -0.326 0.440 0.231 0.336 -0.218 0.375 0.381 0.163 0.575 0.552 0.161 0.342 0.083 -0.072 0.199 -0.173 0.206

3 1.00 0.349 0.480 0.511 0.584 0.540 -0.280 -0.173 0.247 0.224 0.314 0.305 0.215 0.056 0.025 -0.260 -0.088 0.051

4 1.00 0.181 -0.092 0.304 0.490 0.011 0.081 0.446 -0.201 0.078 0.113 0.332 0.307 0.130 0.078 -0.078 -0.423

5 1.00 0.728* 0.937**

0.695* 0.366 0.349 0.360 0.831

** 0.781

** 0.676

* 0.670 0.475 -0.108 0.142 0.194 0.033

6 1.00 0.767**

0.615 -0.013 0.006 0.217 0.815**

0.738* 0.810

** 0.481 0.475 -0.232 -0.341 0.275 -0.139

7 1.00 0.741* 0.311 0.313 0.377 0.755

* 0.727

* 0.723

* 0.602 0.363 -0.108 0.036 0.182 -0.161

8 1.00 -0.209 -0.055 0.195 0.376 0.373 0.525 0.252 0.369 -0.050 -0.228 0.245 -0.064

9 1.00 0.776**

0.229 0.420 0.311 0.222 0.589 0.114 -0.225 0.587 0.258 -0.066

10 1.00 -0.169 0.456 0.208 0.349 0.370 0.172 -0.682* 0.075 0.598 -0.090

11 1.00 0.202 0.588 0.034 0.620 0.366 0.548 0.561 -0.472 -0.146

12 1.00 0.862**

0.775**

0.646* 0.556 -0.367 -0.064 0.341 -0.053

13 1.00 0.694* 0.808

* 0.730

* 0.012 0.098 -0.073 -0.267

14 1.00 0.633* 0.662

* -0.427 -0.381 0.391 -0.524

15 1.00 0.750* -0.037 0.309 0.035 -0.340

16 1.00 -0.157 -0.143 0.112 -0.436

17 1.00 0.588 -0.909**

0.001

18 1.00 -0.437 -.233

19 1.00 0.131

20 1.00

* Correlation is significant at the 0.05 level.,

** Correlation is significant at the 0.01 level.

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Table 5.13 Correlation matrix among soil 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 Zn

DTPA

Zn

Plant

Fe Fe

DTPA

Fe

Plant

Cu Cu

DTPA

Cu

Plant

Mn Mn

DTPA

Mn

Plant 1 1.00 0.240 -0.411 0.131 0.621 0.200 -0.228 -0.457 0.245 0.441 0.312 -0.006 0.537 0.542 -0.148 0.477 0.157 0.487 0.324 0.034

2 1.00 -0.312 -0.161 0.129 0.326 -0.298 -0.330 0.014 0.028 0.054 0.328 0.015 0.486 -0.308 0.094 -0.087 0.268 0.062 0.301

3 1.00 0.080 -0.254 -0.187 0.414 0.013 -0.296 -0.431 -0.427 -0.643* 0.171 0.051 -0.078 -0.095 0.053 -0.154 0.048 0.132

4 1.00 0.287 0.271 -0.064 -0.311 0.688* 0.764* 0.680* 0.329 0.207 0.074 0.586 0.805** 0.977** 0.696* 0.743 0.685*

5 1.00 0.026 0.205 -0.415 0.371 0.207 0.447 0.076 0.812** 0.518 0.489 0.372 0.339 0.273 0.581 0.125

6 1.00 -0.812** -0.770** -0.116 0.441 0.119 0.193 -0.332 -0.198 -0.098 0.137 0.120 0.421 -0.159 0.109

7 1.00 0.341 0.047 -0.520 -0.112 -0.420 0.539 0.362 0.344 -0.071 0.056 -0.452 0.244 -0.087

8 1.00 0.141 -0.243 -0.091 0.178 -0.188 -0.260 -0.071 -0.217 -0.209 -0.355 -0.103 -0.104

9 1.00 0.728* 0.937** 0.695* 0.366 0.349 0.360 0.831** 0.781** 0.676* 0.670* 0.475

10 1.00 0.767** 0.615 -0.013 0.006 0.217 0.815** 0.738* 0.810** 0.481 0.475

11 1.00 0.741* 0.311* 0.313 0.377 0.755* 0.727* 0.723* 0.602 0.363

12 1.00 -0.209 -0.055 0.195 0.376 0.373 0.525 0.252 0.369

13 1.00 0.776** 0.229 0.420 0.311 0.222 0.589 0.114

14 1.00 -0.169 0.456 0.208 0.349 0.370 0.172

15 1.00 0.202 0.588 0.034 0.620 0.366

16 1.00 0.862** 0.775** 0.646* 0.556

17 1.00 0.694* 0.808** 0.730*

18 1.00 0.633 0.662*

19 1.00 0.750*

20 1.00

* Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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Table 5.14 Correlation matrix of soil physical properties, soil macronutrients and microclimatic conditions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 pH EC OC% CEC WHC Sand Silt Clay

N (av)

N(total)

%

P2O5

%

K2O % Altitude Oil % Temperat

ure

Plant

height

1 1.00 0.240 -0.411 0.131 0.621 0.200 -0.228 -0.457 0.424 0.083 0.040 -0.208 -0.697* -0.176 0.742

* -0.031

2 1.00 -0.312 -0.161 0.129 0.326 -0.298 -0.330 -0.002 -0.385 -0.228 0.325 -0.692* -0.488 0.726

* -0.218

3 1.00 0.080 -0.254 -0.187 0.414 0.013 0.282 0.325 -0.539 -0.302 0.285 0.279 -0.503 -0.258

4 1.00 0.287 0.271 -0.064 -0.311 0.508 0.501 0.323 0.036 0.087 0.31 -0.168 -0.367

5 1.00 0.026 0.205 -0.415 0.303 0.032 -0.128 0.234 -0.131 0.487 0.293 -0.183

6 1.00 -0.812**

-0.770**

0.319 -0.336 -0.031 -0.059 -0.167 -0.598 0.141 -0.590

7 1.00 0.341 0.013 0.533 -0.135 0.103 0.240 0.741* -0.322 0.322

8 1.00 -0.628* 0.015 0.157 0.009 0.333 0.318 -0.200 0.638

*

9 1.00 0.588 0.093 -0.141 -0.342 -0.133 0.092 -0.516

10 1.00 0.278 -0.326 -0.072 0.199 -0.173 0.206

11 1.00 0.349 0.025 -0.260 -0.088 0.051

12 1.00 0.130 0.078 -0.078 -0.423

13 1.00 0.588 -0.909**

0.001

14 1.00 -0.437 0.233

15 1.00 0.131

16 1.00 * Correlation is significant at the 0.05 level.

** Correlation is significant at the 0.01 level.

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5.5. Conclusions

Aerial parts of ten samples of Craniotome furcata family Lamiaceae, collected

from different locations in Central Himalayas, India was analyzed by GC and GC/MS

for their essential oil composition. Cluster analysis was done to differentiate plants

collected from different locations on the basis of their main constituents. 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 classified wild C. furcata in to four groups on the basis of major

constituents. The genus is classified into four chemotypes as follows:

Chemotype I: Binsar and Munsiyari (δ-Elemene and germacrene D)

Chemotype II: Jeolikot, Mussoorie, Mukteshwar and Kilbury (Germacrene D)

Chemotype III: Rushi and Nainital (α-Bisabolol oxide A, α-cadinol and

germacrene D-4-ol)

Chemotype IV: Bhowali and Ramgarh (α-Zinziberene, α-muurolol and

germacrene D)

Correlation among major constituents

δ-Elemene was positively correlated with germacrene D. Germacrene D-4-ol

was positively correlated with α-cadinol and α-bisabolol oxide A while α-cadinol

correlated with α-bisabolol oxide A

Effect of macronutrient and micronutrients on essential oil composition

Correlation analysis revealed that micronutrients in soil and plant affected

essential oil composition. Available nitrogen was negatively correlated with δ-elemene.

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Total and available zinc, available copper and total manganese in soil was positively

correlated with α-bisabolol oxide A, suggesting the role of nitrogen, zinc, copper and

iron in their biosynthesis in C. furcata.

Effect of plant characteristics and microclimatic conditions on essential oil

composition

Altitude was positively correlated with plant height with δ-elemene and

negatively correlated with α-muurolol.

Thus, it can be suggested that essential oil composition of Craniotome furcata

was affected by variation in soil properties and microclimatic conditions. The four

chemotypes were detected on the basis of germacrene D content. At higher altitude,

more germacrene D was synthesized in C. furcata. Nitrogen and Iron in soil

negatively affect synthesis of δ-elemene while zinc, copper and manganese in soil

positively affect the synthesis of α-bisabolol oxide A in C. furcata. The percentage of

δ-elemene was found to be more in taller plants.

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