AN ABSTRACT OF THE THESIS OF
Xinfeng Liu for the degree of Master of Science in Wood Science presented
on March 17, 2009.
Title: Analysis and Comparison of Essential Oil Components Extracted from the
Heartwoods of Leyland Cypress, Alaska Yellow Cedar, and Monterey Cypress.
Abstract approved: ___________________________
Joseph J. Karchesy
The essential oil components of cedar heartwoods play an important role in the
durability of these trees. Yet, the composition of these oils and identity of many
of the compounds remains unknown, or incompletely know for some
commercially important cedar heartwoods. The essential oil extracts of Alaska
Yellow Cedar (Chamaecyparis nootkatensis), Monterey Cypress (Cupressus
macrocarpa), and Leyland Cypress (xCupressoparis leylandii ) which is an
intergenetic hybrid of the first two species is such a case. GC-MS analyses were
carried out on the heartwood essential oil extracts of these three species in
order to determine the chemical composition of each essential oil and to
compare the composition of the intergenetic hybrid (Leyland Cypress) with its
parent species. Analyses were carried out on both steam distilled (6 and 12 hour)
and solvent extracted oils. For example in the 6 hour distilled oils, Carvacrol was
the major component of all three oils, 27% (Alaska Cedar), 67% (Leyland Cypress)
and 82% (Monterey Cypress). Terpinen-4-ol and nootkatin were also found in all
three oils, but in lesser amounts (3-6%). Only the oils from Alaska cedar and
Leyland cypress contained the eremophilane sesquiterpenoids valencene,
nootkatene, epinootkatol, nootkatol, 13-hydroxy valencene and nootkatone.
This family of compounds was completely absent in Monterey cypress oil.
Carvacrol, nootkatin and the eremophlianes make up 68-90% of the oils of all
three species. Similar results were obtained with the 12 hour steam distilled and
solvent extracts. As an intergenetic species, the components in the essential oil
from Leyland Cypress show an intermediate amount of the above compounds
when compared to its parents. However, the amount of hinokitiol (a
monoterpene tropolone) in Leyland cypress surpassed both of its parents. It is
expected that Leyland Cypress should have similar antifungal and insect
resistant properties as Alaska cedar according to the essential oil components,
which needs to be confirmed by future studies. Leyland cypress also shows
promise for a source of important biobased chemicals such as nootkatone and
its related compounds given the fast growth of this tree.
©Copyright by Xinfeng Liu
March 17, 2009
All Rights Reserved
Analysis and Comparison of Essential Oil Components Extracted from the
Heartwoods of Leyland Cypress, Alaska Yellow Cedar, and Monterey Cypress.
by Xinfeng Liu
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented March 17, 2009
Commencement June 2009
Master of Science thesis of Xinfeng Liu presented on March 17, 2009.
APPROVED:
________________________________________
Major Professor, representing Wood Science and Engineering
________________________________________
Head of the Wood Science and Engineering
________________________________________
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of
my thesis to any reader upon request.
__________________________________
Xinfeng Liu, Author
__
ACKNOWLEDGEMENTS
First of all, all my thanks cannot express my appreciation of Dr. Joe Karchesy
enough who is my major professor. It is your supports that bring me here in
Oregon State University. It is your encouragement that makes me heading to the
higher level of my education. And it is your love that makes me feel home here
in U.S. all myself alone. Last but not least, it is your guide that makes me who I
am today not only for study but also for life.
Also, my thanks should go to Dr. Hong Liu who is my minor Professor and Dr.
Kaichang Li for being my committee member. I learned a lot from you in the
classes and thanks for your time and patience for helping me reviewing my
thesis.
I would like to thank Dr. Rick Kelsey from USDA Forest Service for helping me
with the GC-MS, and Dr. Sheeba Veluthoor for helping me solving all the
problems during the experiments.
Last but not least, I should thank my parents for all those unlimited supports and
love. I love you so much.
TABLE OF CONTENTS
PAGE
Introduction...................................................................................................... 15
Alaska-Cedar.................................................................................................... 1
Monterey cypress ............................................................................................ 4
Leyland cypress ...............................................................................................7
Literature Review .............................................................................................. 13
Alaska cedar .................................................................................................. 13
Monterey Cypress.......................................................................................... 18
Leyland Cypress ............................................................................................. 20
In Summary ................................................................................................... 20
EXPERIMENTALS................................................................................................ 23
General Experimental Procedure ................................................................... 23
Extraction of the Essential Oil ........................................................................ 24
Steam distillation:.......................................................................................... 25
Solvent Extraction.......................................................................................... 27
Spectroscopic analysis ................................................................................... 27
Retention Indices........................................................................................... 28
RESULTS AND DISCUSSION ................................................................................ 29
GC – MS......................................................................................................... 29
Discussion...................................................................................................... 34
Steam distillation ....................................................................................... 34
TABLE OF CONTENTS (Continued)
PAGE
Board vs Wood ........................................................................................... 37
Solvent extraction ...................................................................................... 38
Conclusions....................................................................................................... 40
Bibliography...................................................................................................... 42
Appendix........................................................................................................... 47
LIST OF FIGURES
Figure
Page
1 THE NATIVE RANGE OF ALASKA-CEDAR. ...........................................................3
2 THE NATIVE RANGE OF MONTEREY CYPRESS IN MACROSCOPE ........................ 5
3 THE NATIVE RANGE OF MONTEREY CYPRESS IN DETAILED MAP. .......................6
4 PARTIAL SEQUENCES OF RBCL IN LEYLAND CYPRESS , MONTEREY CYPRESS ,
AND ALASKA CYPRESS .................................................................................. 9
5 SOME OF THE STRUCTURES OF THE COMPOUNDS FOUND INSIDE THE
ESSENTIAL OIL FROM ALASKA CEDAR MONTEREY CYPRESS AND ALASKA
CEDAR. ....................................................................................................... 17
6 STRUCTURE OF TROPOLONE, P-MENTHANE, P-CYMENE AND EREMOPHILANE
................................................................................................................... 22
7 APPARATUS FOR STEAM DISTILLATION ........................................................... 26
8 THE SCHEMATIC STRUCTURE OF P-METHANE, P-CYMENE AND EREMOPHILANE
FAMILY AND EXAMPLES IN THE ESSENTIAL OIL FROM ALASKA CEDAR,
MONTEREY CYPRESS AND LEYLAND CYPRESS. ............................................ 36
9 THE CONVERSION OF NOOTKATOL AND EPI-NOOTKATOL TO NOOTKATENE AND
NOOTKATONE............................................................................................. 38
LIST OF TABLES
Table
Page
1 PERCENTAGE COMPOSITION OF MONOTERPENE HYDROCARBON MIXTURES
OF OILS ISOLATED FROM SOME SAMPLES OF LEYLAND CYPRESS FOLIAGE......... 19
2 THE COMPONENTS OF THE 1ST 6HR DISTILLATION FRACTION FROM ALASKA
CEDAR, LEYLAND CYPRESS AND MONTEREY CYPRESS ANALYZED BY GC-MS ...... 29
3 THE COMPONENTS OF THE 6-12HR DISTILLATION FRACTION FROM ALASKA
CEDAR, LEYLAND CYPRESS AND MONTEREY CYPRESS ANALYZED BY GC-MS ...... 30
4 THE COMPONENTS OF THE 6HR DISTILLATION FRACTION FROM ALASKA
CEDAR FRESH WOOD AND LUMBER ANALYZED BY GC-MS................................. 31
5 THE COMPONENTS OF THE 24HR SOLVENT EXTRACTION FRACTION FROM
ALASKA CEDAR, LEYLAND CYPRESS AND MONTEREY CYPRESS ANALYZED BY
GC-MS............................................................................................................... 32
6 THE COMPONENTS OF THE 24HR AND 24-48 HR SOLVENT EXTRACTION
FRACTION FROM MONTEREY CYPRESS ANALYZED BY GC-MS ............................ 33
LIST OF APPENDIX
Appendix
1
Pages
ALASKA YELLOW CEDAR GAS CHROMATOGRAM FOR 6 HOURS STEAM
DISTILLATION.............................................................................................. 48
2 MONTEREY CYPRESS GAS CHROMATOGRAM FOR 6 HOURS STEAM
DISTILLATION.............................................................................................. 49
3 LEYLAND CYPRESS GAS CHROMATOGRAM FOR 6 HOURS STEAM
DISTILLATION.............................................................................................. 50
4 MONTEREY CYPRESS GAS CHROMATOGRAM FOR 12 HOURS STEAM
DISTILLATION.............................................................................................. 51
5
ALASKA YELLOW CEDAR GAS CHROMATOGRAM FOR 24 HOURS SOLVENT
EXTRACTION............................................................................................... 52
6 MONTEREY CYPRESS GAS CHROMATOGRAM FOR 24 HOURS SOLVENT
EXTRACTION............................................................................................... 53
7 LEYLAND CYPRESS GAS CHROMATOGRAM FOR 24 HOURS SOLVENT
EXTRACTION............................................................................................... 54
8 MONTEREY CYPRESS GAS CHROMATOGRAM FOR 48 HOURS SOLVENT
EXTRACTION............................................................................................... 55
Analysis and comparison of essential oil extracted from Leyland Cypress Alaska
Yellow Cedar Monterey Cypress
Introduction
Alaska-Cedar
Alaska-cedar (Chamaecyparis nootkatensis), also known as Alaska yellow-cedar,
yellow-cedar, Alaska cypress, and Nootka cypress, is an important timber
species of northwestern America.Alaska-cedar grows from northern California to
Prince William Sound, AK. Except for a few isolated stands, it is found within 160
km of the Pacific coast. Isolated stands in the Siskiyou Mountains, CA, near the
Oregon border mark its southern limit [1]. In Oregon and Washington,
Alaska-cedar grows in the Cascade Range and Olympic Mountains; scattered
populations are found in the Coast Ranges and in the Aldrich Mountains of
central Oregon. In British Columbia and north to Wells Bay in Prince William
Sound, AK, it grows in a narrow strip on the islands and coastal mainland. An
exception in British Columbia is an isolated stand near Slocan Lake about 720 km
inland. [2]
Alaska-cedar is notable within the cypress family for its tolerance of cool and
wet conditions. The climate of its natural range is cool and humid. Climatic
conditions at elevations where Alaska-cedar grows in the Cascade Range of
Washington are somewhat comparable to those at sea level in coastal Alaska.
Growing seasons are short.
2
Alaska-cedar is relatively free from damage by insects. No infestations of
defoliating insects are known. [1] The wood, however, is very durable and
resistant to fungal attack, partly because of naturally occurring
chemicals-nootkatin, chamic acid, and chaminic acid-in the heartwood that
inhibit fungal growth at low concentrations. [3] Certain "black-stain" fungi are
capable of degrading nootkatin, thereby increasing the susceptibility of the
heartwood to decay [4]. Living trees often attain great age, and over time
heart-rotting fungi cause considerable loss and defect in standing trees [5]. Since
at least 1880, Alaska-cedar has suffered advancing decline and mortality on
more than 100 000 ha (247,000 acres) of bog and semibog land in southeast
Alaska. Abiotic factors appear to be responsible, but the primary cause remains
unknown [4].
Special attributes of Alaska-cedar wood include durability, freedom from
splitting and checking, resistance to acid, smooth-wearing qualities, and
excellent characteristics for milling [6, 7]. It is suitable for boatbuilding, utility
poles, heavy flooring, framing, bridge and dock decking, marine piling, window
boxes, stadium seats, water and chemical tanks, cooling towers, bedding for
heavy machinery, furniture, patterns, molding, sash, doors, paneling, toys,
musical instruments, and carving. The wood is highly regarded in Japan, and
most high-quality logs are exported.
3
Figure 1 The native range of Alaska-cedar. [2]
4
Monterey cypress
Monterey cypress (Cupressus macrocarpa) is a medium-sized evergreen tree,
which often becomes irregular and flat-topped as a result of the strong winds
that are typical of its native area. It grows to heights of around 10-25 m, and its
trunk diameter reaches 0.6 m, rarely up to 1 m or more
Monterey cypress thrives near the sea on the west coast of the United States,
where it is native to the Monterey Bay, California (Figure 2, 3), in two groves, at
Cypress Point and Point Lobos. [8] It is also found in British and New Zealand. It
is restricted to a narrow coastal strip on rocky cliffs, slopes and headlands,
forming pure stands or associated with Pinus radiata, in loam or sand over
granitic rocks or in rock crevices. The climate is of the Mediterranean type, with
dry summers cooled by frequent fog, within reach of ocean salt sprays, and
winter rain. [9]
Cypresses may be infested with aphids, mealybugs, caterpillars, and scale insects.
All can be controlled by washing with soap solution or with appropriate chemical
spray. Monterey cypress is susceptible to coryneum canker fungus, for which
there is no cure. Control of cankers consists chiefly in cutting out and burning
affected parts. Badly infected trees may require complete removal. [10]
5
This species of Cypress has been widely introduced in California and around the
Figure 2 The native range of Monterey cypress in macroscope [11]
6
Figure 3 The native range of Monterey cypress in detailed map. [12]
world, mostly as ornamental tree and to act as wind shelter belts both in
agriculture and horticulture. It is extremely resistant to wind and tolerant of
salty ocean wind and is not easily affected by drought or pests. Several cultivar
forms, notably with yellow-green foliage, have been developed. [9] The timber
was used for fence posts before electric fencing became popular. Sawn logs are
used, by many craftspeople, some boat builders, and small manufacturers, as a
furniture structural material and a decorative wood because of its fine color. It is
7
also a fast, hot burning, albeit sparky (therefore not suited to open fires),
firewood.
Leyland cypress
Leyland cypress (xCupressocyparis leylandii) is a putative, spontaneous hybrid
between Alaska cedar (Chamaecyparis nootkatensis) and Monterey Cypress
(Cupressus macrocarpa) that originated in England in 1888. This tree will grow
18 to 20m tall and 4 to 6m wide. Heights of 20 to 30m are not uncommon.
Leyland cypress was discovered in England by Mr. C. J. Leyland. In the year 1888,
Mr. C. J. Leyland in England planted the seeds that he collected from and Alaska
cypress which was growing near a Monterey cypress. When the seeds became
the seedlings and continued to grow, he found that there are several of them
differed from all the others which are the same as Alaska cypress
morphologically. A couple of decades later, in the year 1911, Mr. C. J. Leyland’s
nephew by chance took some seeds from a Monterey cypress which neighbors
an Alaska cypress. When the seedlings grew up, surprisingly, some of them
showed different growth patterns. However, when the two grown up from
Alaska cypress and Monterey cypress were compared with each other, it is found
that the corns and branches are highly similar for which Mr. C. J. Leyland thought
8
the new species should be a natural crossing of Alaska cypress and Monterey
cypress. And then this specie was named after Mr. C. J. Leyland as Leyland
cypress (xCupressocyparis leylandii). [13]
Since that time, many cultivars have been selected that differ in coloration and
growth habit for use in shelterbelts, hedges, landscape plantings, and Christmas
tree production. Plants will tolerate a wide range of soil types from clay to sand,
acid to alkaline. It grows well in full sun, but tolerates partial shade. Growth is
best when moisture is adequate, but it is also drought tolerant and suitable for
dry sites. Classified hardy to USDA Hardiness Zone 7 (average minimum winter
temperature 0° to 10°F), Leyland cypress is relatively cold hardy and well suited
to plantings throughout the southeast. [14]
It is reported that in New Zealand cypress is the third most utilized exotic species
which is about 20000 m3 cut per year. Monterey cypress takes an essential part
of it and Leyland cypress raises more interests recently because of its fast growth
rate, appearance and higher property as a framing structure.
With the development of the modern genetic technology, many classifications of
plant species are questioned. The scientists used to classify species according to
their morphological and anatomical characters which is not so precise that
sometimes they would make mistakes. There is a lot of important information
9
that could not be seen by eyes such as DNA which is the carrier of the genetic
information that decide how the descendants looks like and what kind of
properties does it have.
Figure 4 Partial sequences of rbcL in Leyland cypress (LC, XCupressocyparis
leylandii), Monterey cypress (MC, Cupressus macrocarpa), and Alaska cypress
(AC, Chamaecyparis nootkatensis) [13]
So in order to confirm that Leyland cypress is the real descendants of Alaska
yellow cedar and Monterey cypress, R.P. Adam and Naoko Yamaguchi et al. did
10
research to find the molecular evidence to support that Alaska yellow cedar and
Monterey cypress is the parents of Leyland cypress. In Naoko Yamaguchi’s study,
gene rbcL from chloroplast and 18S-rDNA are extracted from two different
samples of Leyland cypress. The partial sequences of each gene were compared
with Alaska cedar and Monterey cypress and the molecular evidence shows that
Leyland cypress is the real descendant of Alaska yellow cedar and Monterey
cypress (Figure 4). [15] Later in Adams’ research, a total of 25 samples of Leyland
cypress were analyzed by DNA fingerprinting (RAPDs) AND Inter-simple
Sequence Repeats (ISSRs) techniques along with Alaska cedar and Monterey
cypress. The results indicate that Leyland cypress were intermediate between
the putative parental species. [16]
While the question whether Leyland cypress is the descendant of Alaska cedar
and Monterey cypress has been solved, another question, which genera should
Alaska cedar belongs to, has been raised. This has been a problem for a long
time which in result that Alaska cedar has been put into four different genera
which are Cupressus, Chamaecyparis, Callitropsis, and Xanthocyparis.
In 1824, Don first put Alaska cedar into Cupressus, which was corrected
according to Spach’s description of Chamaecyparis. [17] Thus, Alaska cedar was
11
named Chamaecyparis nootkatensis. About 30 years later, in the middle 19th
century, Örsted transferred Alaska cedar to Callitropsis by his assumption that
Callitropsis was most closely related to Callitris and Fitzroya. [15] For a long time,
there has no questions been brought up until modern technologies has been
applied to Alaska cedar. Its biochemical and genetic information, which cannot
be seen, was collected during the next more than 100 years. In 2002, a new
species, Xanthocyparis vietnamensis, was found in Vietnam which was closely
related to Alaska cedar. In order to accommodate both species, Xanthocyparis
was created. [18] However, this is not the end of this story. There are still the
evidences needed to prove the placement of these two species is proper based
on their DNA information. In the year 2004, Little et al. pointed out that as
circumscribed by Farjon et al., Xanthocyparis included the type species of
Callitropsis. [15] Based on the rule of the international Code of Botanical
Nomenclature, Callitropsis as the earlier-published name has nomenclatural
priority over Xanthocyparis if that gens includes Cupressus nootkatensis. So Little
et al. there for recognized Xanthocyparis as a synonym of Callitropsis. Finally, the
Alaska cedar is transferred back to Callitropsis, however, this has to be approved
by International Botanical Congress in 2011 before its recognition.
After all, the study of their durability raises the attention of the scientist recent
12
years. The durability of these woods is linked to the bioactive terpenes found in
their heartwoods and it is unknown that how the genetic relationship of these
three trees has affected the terpenes produced in each. The objective of this
work was to compare the heartwood essential oil profiles of each species by
GC-MS.
13
Literature Review
Alaska cedar
The first study of Alaska cedar essential oil in record was done by Clark and
Lucas et al. in 1926 to got the essential oil from distillation of leaf and
heartwood, which α-pinene and limonene were identified while β-pinene,
sabinene and p-Cymene’s existence were suspected. [19] During the cooling of
the distillation Clark and Lucas failed to notice and report the formation of some
crystal, which raises the attention of Duff and Erdtman later in 1954. Then they
fully identified the structure of this crystal which is nootkatin. The
characterization of nootkatin’s chemical structure was also carried out by Duff
and Erdtman according to the X-ray analysis combined with the classic chemistry
method. [20] The separation of the pure nootkatin was also achieved by
Gas-liquid chromatography done by Johnson et al. in 1975. [21] The earlier study
showed that nootkatin which is from Alaska cedar heartwood is a member of
tropolones which has a potential capability of fungicides, which can help explain
the excellent durability of Alaska cedar.
And later, another tropolone was also
discovered in the Alaska cedar’s heartwood essential oil, which was chanootin.
14
Its structure was characterized by Norin in 1964. [22]
Erdtman et al. isolated Carvacrol, chaminic acid, chamic acid and carvacrol
methyl ether later. [23, 24, 25] However, the credit of the identification of the
structures of two acids should be given to Norin. [26] Carvacrol is generally
regarded as an anti-ceptic compound and is more commonly associated with
agricultural products such as thyme. And according to Anderson’s study,
carvacrol also carries the fungicidal properties. [27]
Nootkatone, which is a eudalenoid sesquiterpene ketone, was isolated by
Erdtman et al in 1962 and named for the tree as a new compound. [28] Later
Valence and Nootkatene were isolated. [25] These sesquiterpene compounds
belong to a family known as ermophilanes. Nootkatone was later isolated by
Macleod et al. from peel oil of grapefruit and is better known today as the
essence of grapefruit. [29] Since its discovery in the 60’s last century, the
synthesis of nootkatone has been discussed and studied because of its
application as additives of food and cosmetics. Today it is either commercially
isolated from grapefruit rinds or synthesized from Valence (an orange oil product)
by an oxidation reaction to give nootkatone.
[30] This compound has much
commercial value today and many recent studies have focused on how to
15
biosynthesize this compound in good yield from Valence. [31]
With the development of isolation technology, some of the minor component
inside the essential oil of Alaska cedar has been isolated and identified recently.
Xiong and Kasawneh isolated nootkatol, 13-hydroxy Valence (a new compound)
and Valencence-11, 12-diol from the heartwood extracts. [32, 33]
The biological property of essential oil from Alaska cedar was first study as a
whole, which showed highly desired insecticide and pesticide characters. Then
the components in the Alaska cedar heart wood essential oil was studied
individually and some of them, such as carvacrol, nootkatone, nootkatol,
13-hydroxy-valencene, are effective insecticides and repellents against such
Vectors as ticks, fleas, and mosquitoes. [34, 35]
And according to a recent
study by Manter, Kelsey and Karchesy, Nootkatol also has demonstrated activity
against the Phytophthora ramorum which is responsible for sudden oak death
and other diseases causing the death of other species. [36]
Jaiswal et al. found totarol, a diterpenoid phenol, in the essential oil from the
outer bark of Alaska cedar, which has been shown antibacterial properties to
inhibit the proliferation of Mycobacterium, the organism responsible for
16
tuberculosis. [37] It would be expected that this compound may also be present
in the heartwood, but his has not been verified as yet.
Kelsey et al studied the standing dead Alaska cedar heartwood after 80 years
from their death in Alaska. They have described the variation of nootkatone,
nootkatene, nootkatol, and nookatin in standing dead Alaska cedar trees and
showed that with the depth increasing from the surface to the core, their
concentration are gradually increasing in the snags which is consistent with
defense theory of the heartwood, which is more obvious when the bark was
removed. [38]
However, there have been no studies of the essential oil of Alaska cedar
heartwood by GC-MS which would help us take a deeper look into the essential
oil of Alaska cedar.
17
Figure 5 Some structures reported from Alaska cedar heartwood [3]
18
Monterey Cypress
Zavarin and Anderson reported the existence of nootkatin and β-thujaplicin in
Monterey cypress heartwood, which is examined by paper chromatography. [39]
However, this is only a qualitative analysis. They didn’t give the yield and their
proportion in the essential oil. Neither do they specify other component inside
the essential oil. Manimaran et al isolated 13 compounds from Monterey
cypress corn essential oils which include terpinene-4-ol (19.42%), dinopol
(15.63%), α-pinene (13.58%), and β-pinene (12.16%). [40] Later ten
sesquiterpenes, many with unusual carbon skeletons, were identified in foliage
of Monterey cypress by Cool. (2005). [41] Ait igri et al. extracted and analyzed
the essential oil from Monterey cypress twig and wood. Sabinene, β-pinene, and
myrcene were the main components of the solvent extraction, and β-pinene,
sabinene, myrcene, α-terpinene, terpineol , and carvacrol were the main
components of the steam-distillated oils respectively.[42] The wood was not
further described with regards to weather it was sapwood, heart wood, or a
mixture. One might speculate that it may have only/mostly been compared of
sapwood since twigs were also analyzed. The essential oils were produced by
hydro-distillation and only monoterpenes were noted. The wood oil contained
92% carvacrol and a variety of other monoterpenes such as α-terpineol and
terpineol-4 which is probably biosynthetical precursors to carvacrol. ∂-pinene
19
was also found at 3.2%. However, research only focus on the heartwood has not
been seen so far to describe the heartwood essential oil component, which is
included in this paper.
Table 1 Percentage composition of monoterpene hydrocarbon mixtures of oils
isolated from some samples of Leyland cypress foliage. The relative amount of
all monoterpene hydrocarbons and that of terpinen-4-ol are given with respect
to the total amount of essential oil of these samples. [37]
Sample 16= Green Spire; sample 17= Haggerston Grey; Sample 22= Naylor’s Blue;
Sample 23= Leighton Green
20
Leyland Cypress
Scheffer et al. analyzed Leyland cypress leaf oil by chromatography from
different clones which is shown in Table 1. [43] While there have been a number
of studies on the foliage and twig essential oils for the various cultivators of
Leyland Cypress, there have been no studies on the heartwood essential oils or
its chemical components. Although there are some discussions about Leyland
cypress’s durability, scientists’ opinions are not consistent with each other.
In Summary
In all, there are three distinct structural families of terpene compounds have
been reported so far from the woods of Alaska cedar and Monterey Cypress,
including p-menthane/p-Cymene Monoterpenes, Eremophilane sesquiterpenes
and s (both mono and sesquiterpene). [44] (Figure 6)
Both Alaska cedar and Monterey cypress have shown abundant amounts of
Carvacrol, p-cymene, and associated compounds. α -Terpinolene and
terpenen-4-ol are p-Menthane, which are closely associated compounds.
21
Only Alaska cedar has shown Eremophilane compounds so far such as
nootkatone and the related Valence and its derivatives.
Both Alaska cedar and Monterey cypress have been shown to possess
tropolones, most notably the sesqinterpene tropolone nootkatin. It will be
interesting to see how these families play out in full essential oil analysis of all
three trees and what genetic relationships might be revealed through this
analysis.
22
Figure 6 Structure of tropolone, p-Menthane, p-Cymene and Eremophilane
Tropolone and its moiety
23
EXPERIMENTALS
General Experimental Procedure
Quantification of essential oil was carried out on a Hewlett Packard (HP) 5890
Series II Gas Chromatograph with an Agilent (J & W Scientific Inc.) DB-5 column
(30m 0.25mm, film thickness 0.25μm). With a split of 1:50, the carrier gas,
helium, carries essential oil to a flame ionization detector that connected to the
column. The oven temperature starts at 100℃ for 1 minute and rise to 150℃ at
a rate of 5℃/min, then to 220℃ at 3℃/min, and finally to 240℃ at 5℃/min
and held at that temperature for 22 minutes.
Then is followed the GC-MS analysis which is performed on the same HP 5890
GC machine with a different Agilent column, DB-5MS (30m×0.25mm, film
thickness 0.25μm) which is connected to a mass selective detector. The
temperature program is basically the same except that in the GC-MS analysis, it
only holds for 5 minute when the temperature reaches 240℃. Helium is also the
carrier gas but at a split of 1:10. The identification of the components of
essential oils was made according to the comparison of their spectra with those
24
in Adams Library. [45]
To remove the solvent left in the extraction, a Buchi Rotavapor (R-110) with a 35℃
water bath was used under reduced pressure by water aspirator.
Extraction of the Essential Oil
The essential oil from Alaska yellow cedar is obtained from Yeping’s work. The
Leyland cypress was collected from Sylvan Option nursery, Dillard, OR. And the
Monterey cypress was collected from Bay Boulevard site, Newport, OR. All three
samples are shipped to the lab as big blocks. Then the blocks are debarked and
the sap woods are removed from the heartwood. After that, the heartwood are
chopped into small pieces as 10×15×1mm. The chips are sealed and frozen until
used. And the voucher specimens of Leyland cypress and Monterey cypress was
deposited to the herbarium of OSU ( Accession No: OSC 218236 for X
Cupressocyparis leylandii and OSC 218244 for Cupressus macrocarpa).
25
Steam distillation:
Steam distillation of the heartwood chips was carried out in the glass apparatus
shown in Figure 7. Typically 1.5-2.0 Kg of chips were steam distilled for 6 hours
first, which give a yield of 5.718g (AYC), 10.243g (MC) and 10.119g (LC) essential
oils respectively. The second batch lasts for another 12 hours to get a yield of 2-3
ml of essential oil. The 12 hours distillation gave a yield of 2.646g (AYC), 2.204g
(MC) and 1.834g (LC) essential oil. After that the essential oil is sealed and frozen
until further analysis. Then the essential oils are dried with anhydrous sodium
sulfate before the yields are calculated and the density measured.
During the distillation of Alaska Yellow Cedar, some crystals were found in the
essential oil.
26
Figure 7 Apparatus for steam distillation
27
Solvent Extraction
About 200 grams of each samples were extracted with 2000ml of diethyl ether
first for 24 hours and then for another 48 hours with the same batch. The 24
hours and 48 hours were collected respectively, and then filtered before it is
dried with anhydrous sodium sulfate. The essential oil was then recovered by a
rotary evaporator under reduced pressure. After all, the yield of the essential oil
was calculated. The Alaska yellow cedar gives a yield of 6.213g (24 hours) and
3.012g (48 hours) respectively. The Monterey cypress gives a yield of 2.390g (24
hours) and 0.615g (48 hours) respectively. And the Leyland cypress gives a yield
of 2.029g (24 hours) and 0.882g (48 hours) respectively.
Spectroscopic analysis
GC-FID and GC-MS were carried out both on the steam distillation samples and
solvent extraction samples. Of each species, 5.0μl of essential oil was dissolved
in 1.0ml of ethyl acetate to make the test solution. Then 1.0μl was injected into
the GC-FID and GC-MS machine. Afterward, the data was collected and analyzed
in the computer.
28
Retention Indices
10μl essential oil was dissolved in 0.5ml Hexanes first. Then the essential oil
solution was mixed with STD D5442 in a 1:1 ratio to get the RI for the
components which have less than 20 carbons. Then the solution was mixed with
14 component Paraffin mix to get the higher carbon component’s RI in the
essential oil in a 2:1 ratio. The way that retention indices are calculated is
100×Rn+100×[(Rx-Rn)/(Rn+1-Rn)], which Rx is the retention time of the compound
X, Rn is the retention time of the standard peak in front of X, and Rn+1 is the one
right after peak X.
29
RESULTS AND DISCUSSION
GC – MS
Essential oils of the 1st 6hr fraction produced for the heartwoods of Alaska cedar,
Leyland Cypress and Monterey Cypress were analyzed by GC-MS and compared
in Table 2. A total of 19 compounds were identified from the essential oil of
these three species.
Table 2 The components of the 1st 6hr Distillation fraction from Alaska cedar, Leyland
cypress and Monterey cypress analyzed by GC-MS
Components
MW
AI
RT
AYC
LC
MC
1
terpineol-4
1193
7.384
2.49%
4.99%
5.58%
2
alpha-terpineol
1204
7.619
0.97%
0.96%
0.69%
3
carvacrol methyl ether
1250
8.676
1.33%
1.44%
0.90%
4
unknown
1276
9.300
2.84%
Trace
Trace
5
carvacrol
1304
9.940
27.15%
66.97%
81.91%
6
hinokitiol
1494
14.863
Trace
5.00%
0.99%
7
valencene
1513
15.284
2.51%
1.01%
-
8
unknown
1516
15.365
0.51%
Trace
-
9
Thujaplicinol<alpha->
1528
15.810
-
-
0.84%
10
nootkatene
1534
15.902
13.29%
3.93%
-
11
selinene<7-epi-alpha->
1539
16.047
0.90%
0.26%
-
12
murrolol<epi-alpha->
1658
19.693
1.43%
0.29%
-
13
cadinol<alpha->
1671
20.101
1.48%
0.34%
-
14
unknown
1679
20.345
0.96%
0.24%
-
15
epi nootkatol
1716
21.513
3.91%
0.96%
-
16
nootkatol
1731
21.985
5.71%
1.49%
-
17
valencene-13-ol
1784
23.684
5.59%
0.35%
-
18
nootkatone
1825
25.024
12.90%
3.35%
-
19
nootkatin
1974
29.807
3.10%
3.85%
5.86%
Total
87.06%
95.42%
96.78%
154
204
204
Trace when the component is lower than 0.10% on wt of the whole essential oil
AI stands for Retention Index and RT stands for Retention Time
30
After collection of the 1st 6 hour fraction, a second essential oil fraction was
collected after an additional 6 hrs distillation time and analyzed (Table 3). Four
compounds that did not show up in the first 6 hr fraction were found in the
second 6 hr fraction, which are alpha-terpinolene, β-thujaplicin, pygmaein and
intermedeol. However, there is one of those found in the first 6hr fraction was
not found in the second 6hr fraction, which is and α-Thujaplicinol.
Table 3 The components of the 6-12hr Distillation fraction from Alaska cedar,
Leyland cypress and Monterey cypress analyzed by GC-MS
Components
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
alpha-terpinolene
terpineol-4
alpha-terpineol
carvacrol methyl ether
unknown
carvacrol
hinokitiol
valencene
nootkatene
selinene<7-epi-alpha->
pygmaein
murrolol<epi-alpha->
cadinol<alpha->
intermedeol
epi nootkatol
nootkatol
valencene-13-ol
nootkatone
19
nootkatin
MW
154
AI
RT
AYC
LC
MC
1193
1204
1250
1276
1304
1494
1513
1534
1539
1599
1658
1671
1679
1716
1731
1784
1825
4.519
7.36
7.597
8.676
9.286
9.943
14.863
15.284
15.893
16.029
17.947
19.678
20.092
20.345
21.504
21.978
23.679
25.000
0.97%
2.53%
1.30%
8.43%
37.45%
0.94%
2.79%
16.59%
1.64%
1.33%
2.02%
3.72%
5.31%
5.90%
4.02%
Trace
5.45% 1.60%
1.19%
0.11%
1.87% 0.14%
0.32%
75.52% 55.79%
Trace
2.90%
0.98%
4.86% 0.97%
0.27%
1.23%
0.27%
0.32%
0.19%
1.13%
1.79%
0.24%
2.34%
Trace
1974
29.798
3.55%
0.80%
Total
98.49%
97.21% 96.61%
33.51%
Trace when the component is lower than 0.10% on wt of the whole essential oil
AI stands for Retention Index and RT stands for Retention Time
31
A comparison was also made between the 6 hrs distillation for Alaska cedar
heartwood and Alaska cedar heartwood from lumber processed. This was to
estimate any changes in the essential compounds which might occur in lumber
mill processing, age of the tree or source location (Table 4).
Table 4 The components of the 6hr Distillation fraction from Alaska cedar fresh
wood and lumber analyzed by GC-MS
Components
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
delta-3-carene
terpineol-4
alpha-terpineol
carvacrol methyl ether
unknown
carvacrol
hinokitiol
valencene
unknown
nootkatene
selinene<7-epi-alpha->
murrolol<epi-alpha->
cadinol<alpha->
unknown
vetivenene<beta->
epi nootkatol
nootkatol
valencene-13-ol
bisabolenol<beta->
nootkatone
unknown
nootkatin
MW
154
204
204
AI
1008
1174
1186
1241
1276
1298
1494
1496
1516
1517
1520
1640
1652
1679
1694
1716
1731
1767
1777
1806
1902
1959
RT
Board
Wood
4.527 0.84%
7.362 0.92% 2.49%
7.599 0.79% 0.97%
8.654 2.26% 1.33%
9.278 0.97% 2.84%
9.915 12.53% 27.15%
14.48 0.59%
Trace
15.259 2.83% 2.51%
15.337 0.52% 0.51%
15.887 17.29% 13.29%
16.022 1.03% 0.90%
19.671 1.40% 1.43%
20.08 1.63% 1.48%
20.32 0.82% 0.96%
20.845 1.04%
21.513 Trace
3.91%
21.985 Trace
5.71%
23.667 6.42% 5.59%
24.177 0.52%
25.005 9.69% 12.90%
28.879 0.50%
29.777 2.20% 3.10%
Total 64.80% 87.06%
Trace when the component is lower than 0.10% on wt of the whole essential oil.
AI stands for Retention Index and RT stands for Retention Time
32
The first 24 hr of solvent extraction by diethyl ether of the heartwoods were also
compared in Table 4. This was done to identify and constituent which might be
changed because of the conditions in the steam distillation process. In Table 5
the major compounds only are summarized.
Table 5 The components of the 24hr solvent extraction fraction from Alaska
cedar, Leyland cypress and Monterey cypress analyzed by GC-MS
Components
MW
AI
RT
AYC
LC
MC
1
terpineol-4
1197
7.456
0.85%
3.74%
3.90%
2
carvacrol
1308
10.045
10.27%
51.79%
65.74%
3
hinokitiol
1498
14.855
0.72%
3.97%
1.71%
4
valencene
1517
15.406
2.02%
1.50%
Trace
5
nootkatene
1539
16.051
9.45%
1.58%
0.62%
6
epi nootkatol
1720
21.654
0.38%
2.23%
-
7
nootkatol
1735
22.127
0.65%
7.68%
-
8
valencene-13-ol
1789
23.863
4.81%
0.53%
-
9
nootkatone
1833
25.266
11.82%
5.68%
-
1939
28.686
18.31%
-
-
1983
30.066
8.11%
6.99%
16.20%
2008
30.860
1.50%
0.55%
5.36%
Total
68.88%
86.24%
93.54%
10
valencene<11,12
-dihydroxy->
11
nootkatin
12
unknown
230
Trace when the component is lower than 0.10% on wt of the whole essential oil
AI stands for Retention Index and RT stands for Retention Time
33
And then another 24 hour extraction of essential oil from Monterey cypress is
compared with that of the first 24 hour in Table 6. Only 9 major components
which are higher than 0.1% in percentage of the whole oil are compared here
and all of them are in good consistency.
Table 6 The components of the 24hr and 24-48 hr solvent extraction fraction
from Monterey cypress analyzed by GC-MS
Components
1
terpineol-4
2
alpha-terpineol
3 carvacrol methyl ether
4
carvacrol
5
unknown
6
hinokitiol
7
unknown
8
nootkatin
9
unknown
MW
AI
RT
MC 24Hrs
230
1198
1208
1253
1309
1321
1499
1531
1981
2008
7.467
7.703
8.765
10.045
10.250
14.873
15.821
30.003
31.103
Total
3.90%
0.48%
0.46%
65.74%
1.22%
1.71%
0.67%
16.20%
5.36%
95.74%
MC
24-48Hrs
3.88%
0.47%
0.57%
63.83%
1.71%
1.16%
Trace
17.34%
5.31%
94.35%
Trace when the component is lower than 0.10% on wt of the whole essential oil
AI stands for Retention Index and RT stands for Retention Time
34
Discussion
Steam distillation
From Table 2, 6 hrs essential oil fractions, it can be seen that the major
component of all three oils is carvacrol with 27% in Alaska cedar, 67% in Leyland
cypress and 82% in Monterey cypress.
Associated p-methane derivatives
terpineol-4 and alpha-Terpineol were also found in all three oils, but at much
lower levels ranging from 2.5 – 5.6% for Terpineol-4 and 0.97 – 0.69% for
alpha-Terpineol. The amounts of most components in Leyland cypress are
intermediate between his parents Alaska cedar and Monterey cypress except the
hinokitiol (5% in LC, 0.99% in MC and only trace amount in AYC) and carvacrol
methyl ether ( 1.44% in LC, 0.90% in MC and 1.33% in AYC).
In Alaska cedar the eremophilane derivatives make up the largest proportion of
the oil at 44%.
Nootkatene (13.21%) and nootkatone (12.90%) are the two
dominant compounds of this group.
Lesser amounts of valencene (2.51%) and
the alcohols, epi-nootkatol (3.91%), nootkatol (5.71%) and valencene-13-ol
(5.51%) make up the remainder of this group.
The same eremophilane
compounds are seen in Leyland cypress oil, but at lower levels (11.09% total).
Again nootkatene and nootkatone are the two major compounds of this group.
The eremophilane group was essentially absent in Monterey cypress oil. The
tropolones were dominated in all three oils by nootkatin which ranged from 3.1%
in Alaska cedar to 5.86% in Monterey cypress.
Hinokitiol was present in
Leyland cypress at 5% while only a trace to 1% was found in the other two
species.
35
Other compounds, including unknown ones, were generally less than 2% of the
oil, except the unknown compound with MW 154 in Alaska cedar which was
detected at 2.84%.
A tentative MS analysis suggested it might be
meth-1-en-9-o1, but this needs to be confirmed.
A second 6 hrs steam distillation (6 – 12 hrs fraction ) after collection of the first
6 hrs, is shown in Table 3. Again carvacrol is the dominant compound ranging
from 37.45% to 75.52% of the oils collected for the three species. However the
nootkatin level rose significantly in the Monterey cypress oil to 33.51%, while it
remained much less in the other two oils.
In summary the steam distilled oils were dominated by carvacrol, the
eremophilane derivatives and nootkatin for Alaska cedar and Leyland cypress.
Carvacrol is well documented for its antifungal, insecticidal, and antibacterial
properties.
Nootkatone and nootkatol are well documented for their
acaricidial, insecticidal, and repellent properties. Nootkatin and hinokitiol are
well known metal chelators with significant antifungal properties. These series
of compounds explain the durability of these heartwoods and present a
potential source of biobased chemicals. Monterey cypress is dominated by
carvacrol and nootkatin as its basis for heartwood durability.
36
Figure 8 The schematic structure of p-Methane, p-Cymene and Eremophilane
family and examples in the essential oil from Alaska cedar, Monterey cypress and
Leyland cypress.
The p-methane derivatives, terpineol-4 and alpha-Terpeniol (and perhaps the
unknown compound 4, mw 154) are believed to be biosynthetic precoursors to
the p-cymene compound, carvacrol. (Some of the structure shown in Figure 8)
37
Board vs Wood
In comparing the essential oil content of the fresh wood vs. processed lumber
scraps we can see the following general aspects.
about the same in each, more or less.
Many of the compounds are
However, the appearance of
delta-3-carene in the board oil may be due to some amount of sapwood
contained in the boards. In general, in the board oil there is also a lower
amount of the p-methanes. This may be due to the higher volatility of these
compounds and their evaporation during lumber processing. Some difference
in nootkatene, nootkatol, epinootkatol, and nootkatone are seen which
tentatively might be expected and explaind as follows. The percentage of
nootkatene goes up in the processed boards while the two alcohols are reduced
to trace amounts. Conifer heartwoods are known to be acidic due to mainly
the presence of acidic acid which is liberated from the acetyl groups of
hemicelluloses in the heartwood. Hillis notes that the heartwoods of old trees
can become notably acidic. [46] The acidic acid formation is autocatalytic.
It
has also been noted that the acidity of stored undried samples can also increase.
Other compounds in the heartwood can also contribute to this acidity. These
include, phenols and of course the tropolones. Nootkatol is also a known
precursor to produce nootkatone via an oxidation process. However, the reason
why nootkatone decreased may be that during the drying or processing, the
essential oil evaporated or because of the individual variation, or maybe both,
which is not limited. As shown in Figure 9, the conversion of nootkatol and
epi-nootkatol to nootkatene and nootkatone can readily occur in wood
processing or aging. This proposal needs to be confirmed, but seems quite
plausible for the observations in Table 4.
38
Figure 9 The conversion of nootkatol and epi-nootkatol to nootkatene and
nootkatone
.
Solvent extraction
Table 5 and 6 show the cold solvent extractions of these heartwoods with diethyl
ether.
The compounds present in these extracts parallel those in the steam
distilled oil with two major exceptions. The presence of valencene-11, 12- diol
and an unknown tropolone with AI 2008 and MW 230 are prominent
components that are likely changed under the conditions of steam distillation.
Valencene- 11, 12- diol would be expected to be thermally and acid labile. The
unknown tropolone appears to an isomer of procerin by its mass spectral
fragmentation pattern, but has the wrong retention time.
isolated and identified in future research.
It needs to be
39
There is no essential difference between the 24 hour fraction and the 24-48 hour
fraction but small fluctuation in the amount of each component. While in the
steam distillation, the fluctuation in the amount of each component in Monterey
cypress is more significant.
40
Conclusions
Essential oil from Alaska cedar, Monterey cypress and Leyland cypress was
extracted from the heartwood by both steam distillation and solvent extraction.
18 ingredients are found both in Alaska cedar and Leyland cypress while there
are only 8 found in Monterey cypress from steam distillation of their heartwoods.
The GC-MS analysis of essential oils from two fractions of steam distillation and
solvent extraction respectively shows that the dominant components in these
three species, Alaska cedar, Monterey cypress and Leyland cypress, are carvacrol,
nootkatene, nootkatol, nootkatone and nootkatin, which represent at least
about 60-90% of the total volume of the essential oil in which carvacrol tops all
the others. These compounds are from 3 different families which are p-Cymene,
eremophilane and tropolone.
The comparison of essential oil from Alaska cedar lumber and fresh wood shows
that along with the aging of the wood, the amount of nootkatol and
epi-nootkatol dropped significantly while the amount of nootkatene rises
notably, which indirectly indicates the conversion of nootkatol to nootkatene in
the lumber, which was accelerated by the increase of acidity inside the
heartwood.
More components are detected by solvent extraction when analyzed with the
GC-MS. Due to the insufficient amount of some compounds, only some of them
were characterized. There are 12 in Alaska cedar, 11 in Leyland cypress and only
7 from Monterey cypress. Except two exceptions, valencene-11, 12- diol and an
unknown tropolone with AI 2008 and MW 230, all the others are found in the
41
steam distillation fractions. The absence of valencene-11, 12- diol and the
unknown tropolone are likely changed during steam distillation because of
higher temperature.
Carvacrol is still the dominant ingredient inside the Leyland cypress and
Monterey cypress heartwood essential oil from the solvent extraction while in
Alaska cedar, valencene-11, 12- diol (18.31%) surpassed carvacrol (10.27%) to
became the most abundant component.
Because of the intergenetic feature of Leyland cypress, genetically, Leyland
cypress shows the intermediate characters most of the time. However, Leyland
cypress shows a greater amount of hinokitiol which has antifungal property,
both in steam distillation and solvent extraction.
The unknown peak after nootkatin has a similar structure as procerin according
to the GC-MS, which could be a tropolone. Further research needs to be done to
isolate this compound and identify its structure.
The blank knowledge of essential oils of Leyland cypress and Monterey cypress
are filled although not thoroughly due to several unknown compounds. It gives
us a more clear view when we compare the durability with one or more species
among the three. In the future, the same method could also be used on other
species that are genetically related.
42
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47
Appendix
48
Appendix 1 Alaska yellow cedar gas chromatogram for 6 hours Steam
distillation
49
Appendix 2
Monterey cypress gas chromatogram for 6 hours Steam
distillation
50
Appendix 3
Leyland cypress gas chromatogram for 6 hours Steam distillation
51
Appendix 4 Monterey cypress gas chromatogram for 6-12
12 hours steam
distillation
52
Appendix 5 Alaska yellow cedar gas chromatogram for 24 hours solvent
extraction.
53
Appendix 6 Monterey cypress gas chromatogram for 24 hours solvent
extraction.
54
Appendix 7 Leyland cypress gas chromatogram for 24 hours solvent extraction.
55
Appendix 8 Monterey cypress gas chromatogram for 24-48
48 hours solvent
extraction.