Upload
others
View
13
Download
0
Embed Size (px)
Citation preview
ETHNOBOTANICAL USES, PHYTOCHEMICAL ANALYSIS, BIOACTIVITY AND MOSQUITO
REPELLENCY OF CYPERUS ARTICULATUS L. FROM THE FAMILY CYPERACEAE
FROM THARAKA MERU
BY
KARAMBU E. MURIITHI
156/78601/2009
DEPARTMENT OF CHEMISTRY /
UNIVERSITY OF NAIROBI
A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF MASTER OF SCIENCE IN ENVIRONMENTAL CHEMISTRY, UNIVERSITY OF NAIROBI t , \
DECEMBER 2012
THIS IS MY ORIGINAL WORK AND HAS NEVER BEEN SUBMITTED FOR AWARD OF
ANY DEGREE IN ANY UNIVERSITY.
DECLARATION
KARAVLARAMBU E. MURIITHI DEPARTMENT OF CHEMISTRY
UNIVERSITY OF NAIROBI
THIS THESIS HAS BEEN SUBMITTED FOR EXAMINATION WITH OUR APPROVAL AS UNIVERSITY SUPERVISORS.
^ J o 2- J X X I 13,PROF. JACOB O. MIDIWO DEPARTMENT OF CHEMISTRY UNIVERSITY OF NAIROBI
11\ o i\DR. JOHN M. WANJOHI DEPARTMENT OF CHEMISTRY
\OF NAIROBI
PROF. STEP HEN G. KIAMA’ DEPARTMENT OF VETERINARY ANATOMY & ANIMAL PHYSIOLOGY, UNIVERSITY OF NAIROBI
DR. PETER M. MATHIU DEPARTMENT OF VETERINARY ANATOMY & ANIMAL PHYSIOLOGY UNIVERSITY OF NAIROBI
SPONSERED BY
RISE-AFFNET
11
DEDICATION
This work is dedicated to ray husband. David Kiogora, my son Newton Murithi
brothers and sisters.
I * /
iii
my parents,
ACKNOWLEDGEMENTS
I sincerely thank my supervisors, Prof. Jacob O. Midiwo, Dr. John M. Wanjohi, Prof. Stephen G.
Kiama and Dr. Peter M. Mathiu, who have instructed, guided, supported and encouraged me
throughout my studies. These are the people who have natured my talents to what I am today. 1
thank the University of Nairobi especially the Departments of Chemistry and Veterinary
Anatomy & Animal Physiology (VAP) for providing me with space and time to carry out this
research. My appreciation also goes to the other academic, technical and support staff from the
Departments of Chemistry and Veterinary Anatomy & Animal Physiology, University of
Nairobi, for their assistance in various aspects of my study. 1 also appreciate my colleagues in the
Natural Product Chemistry Research laboratory the Pesticides Research laboratory, Veterinary
Anatomy & Animal Physiology laboratory, and any other masters or Ph.D. students who assisted
me and gave me company. My appreciation also goes to the traditional medicine practitioners
from Meru who provided the ethno-botanical information on C. articulatus L and Mr. Mutonga
who was very instrumental during the plant material collection. 1 greatly thank Dr. Moses Langat
of Surrey University, UK who carried out the GC-MS analysis of my samples and took the pains
to search some reference materials for me. My acknowledgement also goes to Pan African
Chemistry Network (PACN) for sponsoring a Gas Chromatography- mass spectroscopy (GC-
MS) workshop during which I gained skills that were very instrumental in my data analysis. I
would also not forget the facilitators of the workshop, especially Dr. M. Schaefer of Germany
and Professor Gachanja of Jomo Kenyatta University of Agriculture and Technology (JKUAT).
finally I thank and appreciate Regional Initiative in Science and .Education- African Natural
Products Training Network (RISE-AFFNET) Cannergy foundation ofi Newyork for their
financial support they provided for me in all that I needed for my research work.
IV
TABLE OF CONTENTSDECLARATION............................................................................................................................ ii
DEDICATION............................................................................................................................... iii
ACKNOWLEDGEMENTS........................................................................................................... iv
TABLE OF CONTENTS............................................................................................................... v
LIST OF FIGURES...................................................................................................................... vii
LIST OF TABLES....................................................................................................................... viii
LIST OF APPENDICES................................................................................................................ ix
CHAPTER ONE.... ,........................................................................................................................ 1
INTRODUCTION....................................................................................................................11.1 General Introduction...........................................................................................................11.2 Problem statement.............................................................................................................. 31.3 Justification........................................................................................................................ 41.4 Objectives.......................................................................................................................... 4
1.4.1 General objective.................................................................................................... 41.4.2 Specific objectives...................................................................................................4
CHAPTER TWO............................................................................................................................ 6
LITERATURE REVIEW........................................................................................................ 62.1 Botanical information........................................................................................................ 6
2.1.1 Family Cyperaceae (Sedge Family.......................................................................... 62.1.1.1 Cyperus rotundus.....................................................................................................72.1.1.2 Cyperus articulatus L ............................................................................................... 82.1.1.2.3 Cultivation.......................................................................-■.................................. 92.1.1.2.4 Geographical Distribution of C. articulatus L.......................................................... 10
2.2 Ethno-Medicinal Uses of C. articulatus L ........................................................................102.3 Essential oils.....................................................................................................................14
2.3.1 Composition...........................................................................................................152.3.2 Source and Isolation............................................................................................... 152.3.3 Chemical Analysis................................................................................................. 16
2.3.3.1 GC/MS Technique.................................................................................................172.4 Terpenes as constituents of essential oils.........................................................................19
2.4.1 Biosynthesis of terpenes....................................................................................... 202.4.2. Monoterpenes CIO................................................................................................ 232.4 .3 Sesquiterpenes C l5............................................................................................... 242.4.4 Functions and utilities of sesquiterpenes..................................................................242.4.5. Diterpenes C20.....................................................................................................252.4.6 Triterpenes C30................;............. y.... ............. .................................................. 25
v \ 1 1
2.4.7 Tetraterpenes C40..................................................................................................25CHAPTER THREE...................................................................................................................... 27
METHODOLOGY................................................................................................................ 273.1 Ethno Botanical Survey of C. articulatus L from Tharaka Meru.................................... 27
3.1.1 Reconnaissance survey.......................................................................................... 283.1.2 Ethno-Botanical Survey Workshop......................................................................... 29
3.2 Plant Material Collection................................................................................................. 303.3 Plant Extraction................................................................................................................ 303.4 Laboratory Analysis......................................................................................................... 31
3.4.1 General................................................................................................................313.4.2 Column Chromatography.......................................................................................323.4.3 GC-MS..................................................................................................................33
3.5 Identification of the Compounds Using GC-MS............................................................. 343.6 Antibacterial Activity Assays.......................................................................................... 34
3.6.1 Disc Impregnation..................................................................................................353.6.2 Seeding of Nutrient Agar Plates..............................................................................353.6.3 Bacterial Susceptibility Testing.............................................................................. 363.6.4 Minimum Inhibition Concentration (MIC)...............................................................36
3.7 Mosquito Repellent Test.................................................................................................. 373.8 Structure Elucidation........................................................................................................ 37
CHAPTER FOUR.........................................................................................................................39
RESULTS AND DISCUSIONS............................................................................................ 394.1 Ethno Botanical Survey in Tharaka................................................................................. 394.2 Ethno-Medicinal Uses and preparation methods for concoctions of Cyperus articulatus Lfrom Tharaka.......................................................................................................................... 394.3. Characterization of essential oils of C. articulatus L....................................................... 41
4.3.1 Column Chromatography.......................................................................................414.3.2.1 Chemical Analysis of the Compounds from C. articulatus L from Tharaka...............414.3.2.2 Proposed Fragmentation Pattern for Compound 33................................................... 54
4.4 In-vitro Antimicrobial Activities..................................................................................... 564.4.1 Minimum Inhibition Concentration....................................v...................................57
4.5 Mosquito Repellent Test Results..................................................................................... 58CHAPTER FIVE.......................................................................................................................... 60
5.0 CONCLUSIONS AND RECOMMENDATIONS.......................................................... 605.1 Conclusions...................................................................................................................... 605.2 Recommendations............................................................................................................ 62References...............................................................................................................................64
APPENDICES...............................................................................................................................70
t.vy| /
LIST OF FIGURES
Figure 2.1 Cyperus articulatus L .....................................................................................................8
Figure 2.2 Dried tubers of C.articulatus L ......................................................................................9
Figure 2.3 Thymol......................................................................................................................... 19
Figure 3.1 Dry vegetation.... ......................................................................................................... 27
Figure 3.2 Map of Kenya and Tharaka district..............................................................................27
Figure 3.3 TMPs explaining the uses of different plants during the afternoon field visit............ 28
Figure 3.4 TMPs digging up tubers of C.articulatus L .................................................................30
Figure 3.5 GC-MS set up...............................................................................................................33
Figure 4.1 mass spectrum for compound 33..................................................................................55
vn
LIST OF TABLES
Table 2.1 Documented ethno medicinal use of C.articulatus L from various parts of the world ] -5
Table 4.1 Uses of C.articulatus L from Tharaka Meru...............................................................
Table 4.2 Retention Time, Molecular Formula and Corresponding Peak Area % of Maximum f0r
compounds from C. articulatus L from Tharaka......................................................................... 4^
Table 4.3 Anti microbial activities of the 100% CH2C12 crude extract of C.articulatus L froi^Tharaka......................................................................................................................................... 5^
Table 4.4 Number of bites at different periods of exposure........................................................ 5 j
LIST OF PLATES
Plate 4.1 Staphylococcus aureus
Plate 4.2 Streptococcus pneumonia
56
56
t\1
Vll l
list o f a p p e n d ic e s
Appendix 2.GC-MS Results for 100% DCM Extract 71
Appendix 3 Gas Chromatogram for thel00% Crude Extract 73
Appendix 4 Mass Spectrum for Compound 1 74
Appendix 5 Mass Spectrum for Compound 2 75
Appendix 6 Mass Spectrum for Compound 3 76
Appendix 7 Mass Spectrum for Compound 4 77
Appendix 8 Mass Spectrum for Compound 5 78
Appendix 9 Mass Spectrum for Compound 6 79
Appendix 10 Mass Spectrum for Compound7 80
Appendix 11 Mass Spectrum for Compound 8 81
Appendix 12 Mass Spectrum for Compound 9 82
Appendix 13 Mass Spectrum for Compound 10 83
Appendix 14 Mass Spectrum for Compound 11 84
Appendix 15 Mass Spectrum for Compound 12 85
Appendix 16 Mass Spectrum for Compound 13 86
Appendix 17 Mass Spectrum for Compound 14 87
Appendix 18 Mass Spectrum for Compound 15 88
Appendix 19 Mass Spectrum for Compound 16 89
Appendix 20 Mass Spectrum for Compound 17 90
Appendix 21 Mass Spectrum for Compound 18 91
Appendix 22 Mass Spectrum for Compound 19 92
Appendix 23 Mass Spctrum for Compound 20 93
Appendix 24 Mass Spectrum for Compound 21 94
Appendix 25 Mass Spectrum for Compound 22 95
Appendix 26 Mass Spectrum for Compound 23 96
Appendix 27 Mass Spectrum for Compound 24 f • 97\ ,
Appendix 29 Mass Spectrum for Compound 26 I 99
Appendix 30 Mass Spectrum for Compound 27 100
Appendix 1 Questionnaire used for traditional medicine practitioners 68
ix
Appendix 32 Mass Spectrum for Compound 29 102
Appendix 33 Mass Spectrum for Compound 30 103
Appendix 34 Mass Spectrum for Compound 31 104
Appendix 35 Mass Spectrum for Compound 32 105
Appendix 36 Mass Spectrum for Compound 33 106
Appendix 37 Mass Spectrum for Compound 34 107
Appendix 38 Mass Spectrum for Compound 35 108
Appendix 39 Mass Spectrum for Compound 36 109
Appendix 40 Mass Spectrum for Compound 37 110
Appendix 41 Mass Spectrum for Compound 38 111
Appendix 42 Mass Spectrum for Compound 39 112
Appendix 43 Mass Spectrum for Compound 40 113
Appendix 44 Mass Spectrum for Compound 41 114
Appendix 45 Mass Spectrum for Compound 42 115
Appendix 46 Mass Spectrum for Compound 43 116
Appendix 47 Mass Spectrum for Compound 44 117
Appendix 48 Mass Spectrum for Compound 45 118
Appendix 49 Mass Spectrum for Compound 46 119
Appendix 50 Mass Spectrum for Compound 47 120
Appendix 51 Mass Spectrum for Compound 48 121
Appendix 52 Mass Spectrum for Compound 49 122
Appendix 53 Mass Spectrum for Compound 50 123
Appendix 54 Mass Spectrum for Compound 51 124
Appendix 55 Mass Spectrum for Compound 52 125
Appendix 56Mass Spectrum for Compound 53 126
Appendix 57- Mass Spectrum for Compound 54 127
Appendix 58Mass Spectrum for Compound 55 128
Appendix 59 Mass Spectrum for Compound 56 ' > ' 129t
Appendix 60 Mass Spectrum for Compound 57 130
Appendix 61 Mass Spectrum for Compound 58 131
Appendix 31 Mass Spectrum for Compound 28 101
x
Appendix 62 Mass Spectrum for Compound 59 132
LIST OF SCHEMES
Scheme 2.1a Mechanism for Biosynthesis of Terpenes.......
Scheme 2. 2 Sesquiterpene (Ci5 Compounds)......................
Scheme 2.3 Squalene..................................................
Scheme 4.1 Fragmentation pattern for compound 33...........
xi
a b b r e v ia t io n s a n d a c r o n y m s
c c Column Chromatography
Cfu colony forming units
dcm Dichloromethane
dmf Dimethylformade
El Electron Ionization
GC-MS Gas Chromatography Mass Spectrometry
MIC Minimum Inhibition Concentration
MS Mass Spectrometry
NIST National Institute of Science and Technology
PTLC Preparative Thin Layer Chromatography
|TLC Thin Layer Chromatography
b v Ultra Violet
UV-VIS Ultra Violet-Visible Spectroscopy
WHO World Health Organization
>ACN Pan African Chemistry Network
Use Regional Initiative in Science and Education
ffnet African Natural Products Training Network
kuat Jomo Kenyatta University of Agriculture and Technology
Xll
ABSTRACT
An ethno botanical survey of C. articulatus L (Ndago) was carried out in Tharaka-Meru district,
Kenya from 8th to 10th October 2010. This study was aimed at determining traditional uses and
procedures used by Traditional medicine practitioners (TMPs) to prepare concoctions of C.
articulatus L for treatment of various ailments. It commenced with a reconnaissance survey
where the researchers met with the TMPs and were briefed on the ethno-botanical uses of C.
articulatus L. At the meeting a one day workshop was arranged and a date was fixed. The
workshop was attended by thirty traditional medicine practitioners and five scientists from the
University of Nairobi. During the workshop the TMPs were issued with questionnaires from
which data was collected and analyzed. The data collected from the questionnaires indicated that
all the TMPs were using C. articulatus L for treatment of typhoid fever, stomachache, headaches,
blisters, wounds, skin rush, abdominal pains; cough, as perfume, as a mouth freshener and insect
repellent. The extinction of the plant was established. The results of this survey were
documented for further reference. The root tubers of C. articulatus L were subjected to extraction
with the following solvent systems; 100% CH2C12. 1:1CH2C12/CH30H, 5% H20/CH30H. The
resultant crude extract of 100% CH2C12 was subjected to a combination of chromatographic
techniques including column chromatography (CC) and preparative thin layer
chromatography(PTLC) for the isolation of compounds. Spectroscopic analysis including UV
and GC- MS were done to determine the structures of the compounds. A total of 59 compounds
were identified, of which 48 (82.76%) were terpenes. Amongst the terpenes were 27
sesquiterpenes (45.76%), 20 monoterpenes (33.90%) 1 triterpene (1.69%), apd 11 non-terpenes
(18.64 %). The major sesquiterpene identified in this essential )pil was a cubenene.
xm
This made it unique amongst all the other oils extracted from C. articulatus L from other parts of
the world (Nigeria, Brazil, Japan. Taiwan, Thailand, Hawaii, and Philippines). The 100% CH 2
Cl 2 crude extract was subjected to anti-bacterial tests against Staphylococcus aureus,
Streptococcus pneumoniae and Salmonella typhi bacterial strains. The inhibition zones were
taken and averaged and the extracts showed activity for the three bacterial strains. The undiluted
crude extract of 100% C.articulatus L showed inhibition against the growth of the micro
organisms tested. The 100% CH2CI2 crude extract was repellent against the mosquito Aedes
egyptii. The above results supported the claims by the traditional medicine practitioners that
C. articulatus L could treat skin lashes and repel the mosquitoes Aedes egyptii.
)l/
XIV
CHAPTER ONE
INTRODUCTION
1.1 General Introduction
Absence of modernized socio-economic and public healthcare systems reinforces
reliance of rural and lower-income urban populations on the use of traditional
medicinal-herbs and plants as complementary aids to routine pharmaceutical market
products (Muhammad et al., 2011). Plants and other organisms are the most efficient
tools for synthesis, capable of making a diversity of secondary metabolite organic
molecules (natural products) that have complex structures with a variety of physical,
chemical, and biological properties. Some of these products are complex and may not
be easily synthesized in the laboratory or are easily accessible from nature. For
example, the anticancer drug (vincristinel and vinblastine 2) extracted from periwinkle,
Catharantus roseus, are among the most widely used natural products in the
pharmaceutical industry. These products have not been challenged by any hew synthetic
substitutes and almost half of the available cancer chemotherapeutic drugs are of plant
origin (Kinghorn et al., 1999). Similarly, 25% of all anti-malarial drugs are endemic to
plants, in particular the commonly known quinine which was obtained from Cinchona
species from South America. The currently recommended artemesinin isolated from
Artemisia annua. Today, there is a worldwide emphasis and research on herbal drugs,
where majority of the studies are based on plants that have been traditionally used or
claimed to have potential therapeutic properties!WHO "2003). Presently, about 200( ' '\ /
plant-derived chemical compounds are being used as drugs or as agents for improving
human health (Farnsworth et al., 1985). It was estimated that the booming trade in
herbal medicine will have reached approximately US$500 billion by the year 2000
World Health Organization (WHO 2003) and governments in most developing
countries are campaigning for the promotion and integration of herbal remedies in
healthcare, as supplementary contribution to modern medical facilities especially in
rural areas where medical care is too expensive for some people. Except for few anti-
malarial drugs, all the other commonly used anti-malarial molecules are based upon
plant-derived compounds (Geoffrey, 1996). Africa has a rich tradition of plant use, an
immense range of climates, cultures and species and has the human and natural
resources to become an even greater producer of natural plant products. The
pharmaceutical potential of African medicinal plants are immense (Rukangira., 2001).
In the traditional set-up, there have been a number of concoctions of plant origin for the
bacterial infection. Many people in developing countries especially in the sub-Saharan
Africa depend on crude extracts from plants for treatment of diseases such as malaria,
cancer, allergies and AIDS (Abram et al., 1990). Kenya is endowed with vast resources
of medicinal and aromatic plants of which C. articulatus L is one. Natural treatments
and alternative medicine can serve to complement more traditional therapies though
some alternative approaches have questions regarding their efficacy and safety. The
main aim of this research was to study the phytochemistry of rhizomes of C. articulatus
L (Ndago) growing in Meru- Kenya. C. articulatus L has been used in the ethno-
medicine of the Ameru for various purposes such as treatment of cough, fever, malaria,
fungal infection, arthritis and abdominal pains during menstrual periods, surface skin1 '
infections, boils and wounds. It has also been used as ajmosquito repellent, perfume and
as a mouth freshener. Most people from the larger Meru community have used Ndago2
in one way or another. It was popularly known in the Ameru tradition as a wonder drug.
The popular perfume of Ndago was used on special occasions in the Ameru community
, for example it was given to the newly circumcised young men and women of their age
who were being prepared for marriage to apply shortly before the commencement of
their popular dance for lovers (Bandago), meaning a sweet lover who has the sweet
scent (smell) of Ndago. Repellency is known to play an important role in preventing the
vector borne diseases by reducing man-vector contact. Synthetic chemicals and
insecticides used for control of vectors are causing irreversible damage to the eco
system, as some of them are non-degradable in nature. Some repellents of synthetic
origin may cause skin irritation and affect the dermis. Majority of commercial repellents
are prepared by using chemicals that have been reported to be unsafe for public use.
Due to unpleasant smell, oily feeling to some users and potential toxicity some people
prefer to use natural insect repellent products. Repellents of plant origin do not pose
hazards of toxicity to human and domestic animals and are easily biodegradable.
Natural products are safe for human when compared to that of synthetic compound (Das
et ah, 2003j
1.2 Problem statement
Tharaka district in Kenya is situated in the semi arid parts of the larger Meru region. By
the time of this study there was not a single tarmac road in the whole district and
medical services were not easily accessible. The nearest well equipped public hospital isn
Meru general hospital situated in Meru central district in Meru county .The othert :
hospitals are Nkubu Consolata and Chogoria PCEA mission hospitals that are situated
3
in neighboring districts and are too expensive for most poor people of Tharaka district.
Traditional medicines practitioners (TMPs) were the most accessible and affordable, as
they even accepted payment in kind (chicken, goats and cows). C. articulatus L is one
of the most widely used medicinal plant by the TMPs for the treatment of malaria,
fever, typhoid, cough, common cold, flu, pneumonia, abdominal pains, fungal infection,
skin rush , wounds, as mouth freshener and as a perfume.
1.3 Justification
Several plants are traditionally used in Tharaka for treatment of typhoid, abdominal
pains, stomachache, common cold, tlu, fever, malaria, skin rush, wounds, blisters,
swollen breasts and fungal infections. C. articulatus L is one of these most popular
plants used traditionally by Ameru in Tharaka and the neighboring Tigania, Igembe,
Chogoria, Mwimbi, Chuka and Iment communities of Meru for the treatment of the
above named diseases, it is also used as mouth freshener, perfume and as mosquito
repellent hence the need to carry out a research on it to establish the scientific basis of
its popular use.
1.4 Objectives
1.4.1 General objective
To document the ethno-botanical uses, characterize the components, determine the
antimicrobial and mosquito repellency potency of C.articulatus L from Tharaka Kenya.
1.4.2 Specific objectives•, *%
1. To conduct an ethno botanical survey of C. articulates L in Tharaka Meru.
2. To extract root tubers of C. articulatus L using different organic solvents systems.
4
3. To determine the components of the extract using chromatographic and spectroscopic
methods.
4. I o carry out anti-bacterial assays on the extract.
5. To carry out mosquito repellency test on the extract.
f\\
i/
5
CHAPTER TWO
LITERATURE REVIEW
2.1 Botanical information
The following ethno-botanical information was used to classify C. articulatus L from
Tharaka.
Kingdom Plantae - Plants
Subkingdom Tracheobionta - Vascular plants
Super division Spermatophyta - Seed plants
Division Magnoliophyta - Flowering plants
Class Liliopsida -Monocotyledons
Subclass Commelinidae
Order Cyperales
Family Cyperaceae - Sedge family
Genus Cyperus L. - flatsedge
Species Cyperus articulatus L. - jointed flatsedge
2.1.1 Family Cyperaceae (Sedge Family)
The plants in the family Cyperaceae grow in wet areas along rivers, ponds or swamps.
1 heir stems are usually solid and three-angled (those edges - you may have to slice it
toward the base to really see it). The leaves, when present, are slender but with a
substantial stem clasping basal sheath with fused edges. The flowers (or florets in this
case) are clustered in spikelets. There are usually 3 stamens, and 2 to 4 feathery stigmas\ ' '
on the pistil. The family Cyperaceae includes approximately 36 genera and about 128
species of Cyperus (Neville et al„ 1968).6
2.1.1.1 Cyperus rotund usC. rotundus is a traditional medicinal plant appearing among Indian, Chinese and
Japanese natural drugs used against involuntary muscle contraction and stomach
disorders. The rhizome oils of this plant from different countries show compositional
differences, suggesting the existence of phytochemical varieties. The essential oil as
well as solvent extracts of the rhizomes of C. rotundus have been subjected to numerous
studies resulting in the isolation of many terpenoids. Essential oils do not crystallize
(Mesmin et al., 2001). In Amazonian region C. prolixus and C. rotundus were
cultivated mostly in home gardens for medicinal purposes and aromatization of dish
washers. They have been widely utilized for various medicinal purposes, including birth
control and induction of labor and in hallucinogenic preparations (Maria et al., 2008). In
South Africa Cyperus rotundus is a multipurpose plant used in traditional medicine to
treat stomach ailments, wounds, boils and blisters. A number of pharmacological and
biological activities including anti-candida, anti-inflammatory, anti-diabetic, anti
diarrhea, anti-mutagenic, anti-microbial, anti-bacterial, anti-oxidant, anti-pyretic and
analgesic activities have been reported for this plant. Previous phytochemical studies on
C. rotundus revealed the presence of alkaloids, flavonoids, tannins, starch and many
sesquiterpenoids. The observed compositional difference between C. rotundus found in
South Africa and the rest of the world could be due to climactic and environmental
conditions, chemo types, nutritional status of the plants, and other factors, which canI *
influence essential oil composition (Oladipupo et al., 2009). Essential oil from the
tubers of Cyperus rotundus, obtained by steam distillation by Kilani et al, was analyzed7
by GC and GC/MS and a total of 33 compounds were identified, the oil was
characterized by its high content of sesquiterpenes with cyperene (30.9%) being major.
The antibacterial activity of oil from tubers of Cyperus rotundus, showed more
important activity against the bacteria Staphylococcus aureus (Kilani et al., 2005).
2.1.1.2 Cyperus articulatus LC. articulatus L was described in 1753 by Carl Linnaeus. The name is considered as
validly published (Brickell, 2003). C. articulatus L is a type of reed-like tropical grass,
used in Meru for medicinal purposes as earlier mentioned. It is an aromatic herbaceous
species of grass with short rhizomes, thin and resistant roots. It grows in damp, marshy
and flooded areas along the rivers and streams (where it can help to control soil
erosion). It can attain a height of 6 feet as shown in figure 2.2.
Figure 2.1 Cyperus articulatus L
It grows in clumps from dividing rhizomes which are about 1cm in diameter some times\ '\ ••
in a series of two or three, connected by an underground stem (f(ain Tree Nutrition,
8
2006). C. articulatus L often occurs in almost pure stands in tropical and warm
temperate localities that provide permanent water. It is distinguished by its robust,
leafless culms. (Gordon et al., 2006) The tall green stems are fibrous, round, and hollow
at the base with jointed flat edge. Its blackish red tubers are 1 to 3 cm long. This is as
shown in figure 2.3.
Figure 2.2 Dried tubers of C.articulatus L /'
C. articulatus L has an aroma similar to lavender and the aromatic properties are used in
folklore medicine to cause a feeling of warmth in the body which aids in the treatment
of digestive disorders and its sedative effects (Rain tree Nutrition, 2006).
2.1.1.2.3 CultivationC. articulatus L prefers a sunny to half shady site. It grows best in loamy wet soil
(Brickell, 2003).
9
2.1.1.2.4 Geographical Distribution of C. articulatus LC. articulatus L is native to Asia. Africa, Texas, the South East of the US, Florida,
Mexico, Central America and S. America (Brickell, 2003). A number of plants species
that are found in wetland areas are important economic resources for women in
Swaziland. C. articulatus L and Schoenoplectus corymbosus plants are used for making
food mats, sleeping mats, bags, and baskets, hence provide economic livelihood to
many women (Edje, 2006). Cyperacea grown in Egypt have been investigated for their
flavanoids (Habershy et al., 1989). Although native to the Amazon, C. articulatus L
(piri-piri) can be found in many other tropical areas and countries. It occurs alongside
the Nile River in Africa just as it grows alongside the Amazon River in South America
(Taylor, 2001).
2.2 Ethno-Medicinal Uses of C. articulatus L
Ethnopharmacology and natural product drug discovery remains a significant hope in
improving the poor livelihoods of rural communities (Nanyingi et al., 2008). Over
many years plants have been used for drugs and as fragrance materials. The chemical
characterization of rhizomes of C. articulatus L. shows the presence of flavonoids,
saponins, triterpenes, sesquiterpenes and ketones. As some of the diseases treated with
C. articulatus L. (migraines, headaches and according to a personal communication also
epilepsy) concern the nervous system, some pharmacological work has been done to
define its interaction with this system. Decoction of rhizomes of C. articulatus L.•„ “Vt ' \
possesses depressant activity in the central nervous', system (Ngp et al., 2001). C.
articulatus L (Piri-piri) has a long history of use in herbal medicine systems in South
10
America. It is a very common remedy for treating nausea, vomiting, stomach-aches and
intestinal gas throughout the continent. In Peru, piri-piri is considered as an
abortifacient, anticonvulsant and anti-epileptic and treats stomach-ache (Taylor, 2001).
The crude drug prepared from the rhizomes of this plant has been used in traditional
medicine as contraceptive (Helena et al., 2006). It is used for diarrhea, dysentery,
digestive disorders and intestinal infections, intestinal worms, epilepsy, to stop bleeding
(internally and externally) and to heal wounds. In Africa, piri-piri is used for malaria,
toothaches, headaches, diarrhea, indigestion and coughs (Taylor, 2006). C. articulatus L
is popularly known as priprioca in Para State (Brazil). Priprioca has aroused scientific
and economic interest because of the pleasant aroma of the volatile oil obtained from
the plant rhizome. This species has great importance in the local pharmacopoeia of
Brazil. It is mainly used as a contraceptive, a painkiller, and in the treatment of diarrhea.
The volatile part of the priprioca extract (the essential oil obtained by hydro-distillation)
mainly consists of a-pinene, P-pinene, limonene, myrtenol, a-copaene, and
caryophyllene oxide (Lucinewton et al., 2008). In Brazil C. articulatus L is cultivated
and commercialized by small holders for direct market sale, and as a raw material for
the perfumery industries. Anticonvulsant, sedative, antibacterial, and activity on
epilepsy were reported from this plant (Maria et al., 2008). Extracts from rhizomes of
Cyperus articulatus L. (Cyperaceae) used in Africa and Amazonia has been used for
many different ailments including; digestive disorders, menstrual irregularity and has
been used for its sedative properties and anticonvulsant properties in the treatment of\ ' '
epilepsy (Bum et al., 2004). Rhizomes of C. articulatus L. pocesses anticonvulsant
properties in animals and this explains its use as a traditional medicine for epilepsy in11
Africa (Ngo., 2001). In Cameroon qualitative chemical characterization of the total
extract showed that C. articulatus L contains flavonoids, saponins, polyphenols,
tannins, terpenes and sugars. The total extract of the rhizome of C. articulatus L did not
appear to possess either an aesthetic or paralyzing effects. In contrast, spontaneous
motor activity is significantly reduced by the extract. However, C. articulatus L does
not seem to have muscle relaxant effects. When associated with sodium thiopental or
diazepam, the extract facilitates sleep induction, and increases the total sleep time
without any concomitant analgesic effects (Vincent et al., 2000). C. articulatus L has
been used traditionally for the treatment of pain, cough, tlu, common cold, fever,
malaria and typhoid. Intensive research on the plant in relation to a number of ailments
has been carried out in Brazil, S Africa, Cameroon, Nigeria and Peru. Many of its
biological actions are attributed to various sesquiterpenes called cyperones which are
also found in other Cyperus plants in the family. Nyasse et al (1988) have isolated the
sesquiterpenoic ketones, mandassidione, mustakone, corymbolone and The alcohol
corymbolol from Cameroonian grown C. articulatus L. Earlier work on the essential
oils from the Canadian grown C. articulatus L has led to the isolation and
characterization of a bicyclic ketone, cyperotundone (Nyasse et al., 1988). Two of these
compounds called cyperotundone and a-cyperone, have been reported to posses anti-
malarial activities, as well as the ability to inhibit nitric oxide synthesis and
prostaglandin synthetase inhibitor. Aspirin and ibuprofen are prostaglandin synthetase
inhibitors (Taylor 2006). Corymbolone is a sesquiterpenoid keto-alcohol first isolated in{ \
'985, in South America from the rhizomes of CypeYus corymhdsus Rottboll. Some
years later, corymbolone was isolated in Cameroon, from C. articulatus L along with12
another sesquiterpene. Two sesquiterpenes, corymbolone and mustakone, isolated from
the chloroform extract of the rhizomes of C. articulatus L, exhibited significant anti-
plasmodial properties. Mustakone was approximately ten times more active than
corymbolone against Plasmodium falciparum (Rukunga et al., 2008). In 2009 ant-
malaria activity of a water and methanol extract of the same plant was reported by
Rukunga and Muthaura et al 2011. C. articulatus L has several uses in many parts of
the world. Table 2.1 gives a brief summary of its uses around the world and table 2.2
shows compounds reported from the plant from various regions of the world.
Table 2.1 Documented ethno medicinal use of C.articulatus L from various parts of the world
Part/location Documented ethno medicinal use
Rhizome / Africa Used for toothaches, headaches, diarrhea, indigestion, and coughs
Rhizome / Brazil Used as an antivenin for snakebite
Rhizome / Ecuador Mix ground rhizome with water for fever, and influenza,
Rhizome / Guyana Used for stomachache.
Rhizome/ Peru
Used for snakebite: the fresh raw rhizome is chewed fresh and the juice swallowed, then the pulp is put onto the bite after it has bled .Used as a contraceptive. Used as a hair tonic and for baldness and as a hemostat.Juice taken as a nerve tonic; in cases of Stress, nervous and mental disorders. Juice is taken for malignant tumors and throat cancer Juice is taken as an abortifacien Used for dysentery and other severe intestinal infections digestive disorders Used as a rheumatic pain reliever. Used for healing wounds.
Rhizome / USA
Used to control nausea, stomach pain, and gas. Used for headaches, epilepsy, blood in the urine, menstrual irregularity, breast pain, and vaginal discharge. Used for vomiting (2 ml fluid extract). Used as aromatic tonic, and Anthelmintic. (Called andrue), Considered
13
Gently stimulating, warming, diffusive,used as a gastric tonic. Used to soothe the nervous system and increase skin blood circulation. As an anti-emetic and carminative; for digestive disorders, and intestinal gas
Leaves/Guinea Used as a cerebral anti-malarial., Used for wounds and hemorrhages
Adopted from Leslie Taylor 2006
2.3 Essential oils
For several centuries terpenes are known to be components of essential oils (fragrant
oils) from leaves, flowers and fruits of plants. Example are a Pinene, p Pinene
Caryophyllene oxide, Mertenol, thymol and eucalyptol (Zhang 2005). Essential oils are
highly concentrated volatile, aromatic essences of aromatic plants and they have been
known since ancient times to possess biological activity. They have a complex
composition, containing from a few dozen to several hundred constituents, especially
hydrocarbons and oxygenated compounds which are highly odoriferous (Rios & Recio,
2005). All essential oils are principally composed of a class of organic compounds built/
of "isoprene units. "An isoprene unit is a set of five connected carbon atoms with eight
hydrogens attached. Molecules built of isoprene units are all classified as “terpenes”
(Dewick, 2002). Terpenes contained in essential oils are compounds with tiny
molecular structures and very small molecular weights (Smith et al., 2001).
Monoterpenes, with sesquiterpenes, are the main constituents of essential oils. While a
few, such as camphor, occur in a near pure form, most occur as complex mixtures, often
of isomers difficult to separate (Solomons, 1996).
14
2.3.1 Composition
Literature information is scanty on the chemical composition of the oil from C.
articulatus L from East Africa and Kenya. However, research works on the oil
composition of other Cyperus species have been reported (Nureni et al„ 2006). The
chemical composition of the volatile oils of Cyperus rotundus has been extensively
studied and four chemo types from different parts of Asia have been reported. The H-
type from Japan was found to contain a-cyperone (36.6%), P-selinene (18.5%), cyperol
(7.4%) and caryophyllene (6.2%) as the main constituents. The M-type from China,
Hong Kong, Japan, Taiwan and Vietnam had a-cyperone (30.7%), cyperotundone
(19.4%), p-selinene (17.8%), cyperene (7.2%) and cyperol (5.6%) as the main
constituents. The O-type from Japan, Taiwan, Thailand, Hawaii and the Philippines was
characterized by cyperene (30.8%), cyperotundone (13.1%) and P-elemene (5.2%). In
addition, the Hawaiian O-type had cyperotundone (25.0%) and cyperene (20.7%) as the
major compounds. Finally, the K-type, also from Hawaii, was dominated by cyperene
(28.7%), cyperotundone (8.8%), patchoulenyl acetate (8.0%) and sugeonyl acetate
(6.9%) (Oladipupo et al., 2009).
2.3.2 Source and Isolation
Almost all odoriferous plants contain essential oils. Depending on the type of the plant,
various parts of the plant may be used for isolation of essential oils, e.g. fruits, seeds,
buds and flowers, leaves, and stems, roots, bark or wood.-The raw material from whicht • '\ ! '
essential oils are manufactured may be fresh, partially*, dehydrated 'or dried, but flower
15
oils must be fresh. Many methods are used for the isolation of essential oils. These
include;
Distillation-It is the most important method in obtaining essential oils from plants.
Merceration & enfleurage- This is used for obtaining oils from flowers and yields
highly fragrant oils.
Solvent Extraction- This technique is used in order to increase yield of oil, or to
extract products that cannot be obtained by any other processes, this was the method
that was used in this study.
Extraction by cold pressing expression- This is applied only for Citrus oils (Mesmin et
al., 2000).
2.3.3 Chemical Analysis
Many methods have been used for studying the chemical composition of essential oil,
these include; 1R, UV, NMR spectroscopies and gas chromatography. Chemical
analysis of essential oils is generally performed using GC (quantitative analysis) and
GC/MS (qualitative analysis). Gas chromatography has three main advantages over
other analytical methods, it is very rapid, it has very high separation capacity and also
very great sensitivity. These may be the reasons for its great preference in essential oils
analysis. Identification of the main components is carried out by the comparison of
both the GC retention times and MS data against those of the reference standards (with
known source). Analytical conditions and procedures used should carefully be
described. These include; apparatus of oil analysis (make and model number of the\ - ••/
equipment), column type and dimensions, carrier gas flow rate, the temperature
16
programming conditions including injection temperature, detector and column
temperatures, in addition to mass spectra (electronic impact). Sometimes identification
by GC/MS must be confirmed by retention indices on two columns of different polarity
but at a different temperature. Data should thus include essential oils optical rotation,
density and refraction index. On the other hand, compounds which are not easily
separated by GC, and molecules structurally similar like stereo-isomeric compounds of
essential oils are analyzed by 13C NMR (Lahlou., 2004).
2.3.3.1 GC/MS TechniqueWhen a beam of electrons (70eV) from the heated filament in the GC-MS collides with
sample molecules it produces a positively charged ion which is the molecular ion. The
molecular ion peak in this case was [M-H] +. The produced molecular ion undergoes a
series of fragmentation reactions to produce other smaller fragments both positive and
negative. Most fragmentations occurs soon after the molecule is ionized (<10‘6 s) in the
ion source. The positively charged ions are repelled by the magnet and pushed forward
towards the Mass Spectrometer (MS) which in this case was used as the detector. These
fragmentation reactions yield ‘finger print’ (mass spectrum) that are detected by the
MS. Only positive fragments are analyzed by the mass spectrometer therefore the
spectrum shown in the appendices for compounds one to fifty nine consisted of only
positive species. The fragmentation is as a result of unimolecular reactions and is solely
due to the internal energy of the ions. This internal energy is imparted during the
ionization step. The ionization energies of most organic molecules a«e known to be inf\ ! '
the range of 7-10eV while the Ionizing electrons has 70eV and imparts 5-8eV of
internal energy to the molecular ion as it is being formed. The internal energy imparted17
to the ion during formation is more than enough to break bonds (2-4eV) and this causes
further fragmentation. This fragmentation is through real chemical reactions with
defined mechanisms, they are not random. Fragmentation reactions are fairly fast
because ions are present in the ion source region for just a few microseconds (10-6s).
2.3.4 Uses of essential oils in medicine and modern civilization
The use of essential oils is potentially in the pharmacological field and in food
preservation technology for its antimicrobial effects (Ciani et al., 2000). Essential oils
are commercially important as the basis of natural perfumes and also of spices and used
for flavoring purposes in the food industry (Okigbo et al., 2009). Terpenoids which are
components of essential oils display a wide range of biological activities against cancer,
malaria, inflammation, and a variety of infectious diseases ( Zhang et al, 2005). Many
essential oils such as eucalyptus oil and peppermint oil are used as additives in
pharmaceutical preparations. They are used not only for their flavor and fragrant
properties but also for their biological activity (Mesmin et al., 2000). The essential oil
of Thymus vulgaris (Lamiaceae) is the natural source of Thymol a monocyclic phenolic
compound (C ioH uO). It is widely used in medicine for its antimicrobial and antiseptic
action against oral bacteria and wound healing properties. Previous investigations have
reported its antioxidant properties (Archan et al., 2009). Thymol has been reported as
anti-cancer agent, but its anti-cancer mechanism has not yet been fully elucidated
(Dipanwita et al., 201 l).The structure of Thymol is as shown in Figure 2.4 below.
18
Figure 2.3 Thymol
In Austria Thymol isolated from C.arliculatus L is said to be an important additive in
cosmetic products (e.g. mouthwash, bath essences, etc.), several traditionally used
medicines, and also a main ingredient in different spices and herbs (e.g. thyme, oregano,
savory, etc.) which results in the fact that these products are daily consumed in
considerable amounts (Thalhamer et al., 2011). In 2004, Brazil was the 10th largest
essential oil importer ($42 million) in the world. Simultaneously, the country is the
world’s fourth leading essential oil exporter (US$98 million). The domestic cosmetic
and perfumery industry fragments as follows: hair care 25%, fragrance 13%, oral care
10%, bath 10%, skin care 9% and deodorant 9%. The country’s growing sales rate in/
cosmetics (17%) has outstripped everyone (including China), except for Argentina. The
market share of the Brazilian cosmetics segment breaks down into: 69% personal care,
18% cosmetics and 13% perfumes. Brazil has already commercialized a number of
traditional raw materials for the fragrance industry. Some interesting emerging essential
oils of the region include Priprioca (C. articulatus L ) (6th international congress on
perfumery and natural raw materials Grasse-France).
2.4 T erp en es as co n stitu en ts o f essen tia l o ils - ■
\ \Terpenes are small organic hydrocarbon molecules,,they may bfe eyclic or acyclic,
saturated or unsaturated (Smith et al., 2001). Terpenoids, also referred to as terpenes,19
are the largest group of natural compounds. Many terpenes have biological activities
and are used for the treatment of human diseases. The worldwide sales of terpene-based
pharmaceuticals in 2002 were approximately US $12 billion. Among these
pharmaceuticals, the anticancer drug taxol and the ant-malarial drug artimesinin are two
of the most renowned terpene-based drugs. Based on the number of the building blocks,
terpenoids are commonly classified as monoterpenes Cio, sesquiterpenes C 15, diterpenes
C20, sesterterpenes C25, triterpenes C30 ,Carotenoids C40, and polyisoprinoids C>40 (
Zhang et al,. 2005). C |0 and C15 terpenes are the chief constituents of essential oils.
Terpenes are what make essential oils unique in the world of natural substances. The
terpenes are structurally diverse and widely distributed. Most terpenes have been
isolated from plants.
2 .4 .1 B io s y n t h e s is o f t e r p e n e s
The 5 carbon building blocks of all terpenoids are isopentenyl phyrophosphate (IPP)
and dimethylally diphosphate (DMAPP). The complete sequence of the formation of the
IPP from the five carbon unit has not yet been elucidated. DMAPP may be derived from
IPP or may be produced independently (not yet clarified). Combination of DMAPP and
IPP via the enzyme prenyl transferase yields geranyl diphosphate (GPP). Linalyl PP and
neryl PP are isomers of geranyl PP which are likely to be formed from GPP by
ionization to the allylic cation. These three compounds give rise to a range of
monoterpenes (Dewick 2002). Scheme 2.1 gives a proposed mechanism for
biosynthesis of terpenes. - -v
20
electrophylic addition
stereospecific loss of proton
S c h e m e 2 . 1 a M e c h a n is m f o r B io s y n t h e s is o f T e r p e n e s
f\) /
21
CIO Compunds
S c h e m e 2 . 1 b B io s y n t h e s is c o n t in u e d
Addition of a further C5 IPP unit to GPP leads to farnesyl diphosphate (FPP) which is
the sesquiterpene precursor.
f\
I /
22
F P P
C-15 Compounds (sesquiterpenes)
S c h e m e 2 . 2 S e s q u i t e r p e n e ( C )S C o m p o u n d s )
FPP gives rise to linear and cyclic sesquiterpenes. The increased chain length and
additional double bond leads to possible cyclization and a huge range of mono, bi- and
tri-cyclic sesquiterpene structures can result. In some cases the tartially' diphosphate
nerolidyl PP has been implicated as a more immediate precursor than farnesyl PP.
(Dewick. 2002).
2 .4 .2 . M o n o t e r p e n e s C IO
The monoterpenes are isolated by either distillation or extraction and find considerable
industrial use in flavors and perfumes. Combination of DMAPP and IPP via enzyme
prenyl transferase yields geranyl diphosphate (GPP) (Scheme 2.1). This produces
geranyl PP (GPP). Linaryl PP and neryl PP are isomers of geranyl PP. These three byI '\ ••
modest changes give rise to a range of monoterpenes 'found as components of volatile
23
oils used in flavouring and perfumery like camphor, a-pinene, a-phellandrene, P-
phellandrene and P-pinene .(Dewick, 2002).
2 .4 .3 S e s q u i t e r p e n e s C |$
Approximately 5000 sesquiterpenes have been reported. Most appear to be derived from
mevalonic acid. Sesquiterpenes are found in most plants and many fungi accumulate
sesquiterpenes .The biosynthesis is not as well worked out as for monoterpenes, but
farnesyl pyrophosphate appears to be the intermediate in the biosynthesis of almost all
other sesquiterpenes. FPP-synthetase forms Zs-farnesyl pyrophosphate or diphosphate.
An ionization mechanism is involved. Geranyl-OPP is an intermediate, but may exist
only in combination with the enzyme (Stan forth, 2006).
2 .4 .4 F u n c t io n s a n d u t i l i t ie s o f s e s q u it e r p e n e s
Scientists are interested in unearthing the reason why plants, insects, and fungi produce
sesquiterpenoids as secondary metabolites. Some sesquiterpenes play a role as
pheromones (chemicals secreted by animals that influence the behavior and
development of other members of the same species) this means that they are responsible
for communication between individuals of the same species. Often they serve as
attractants, thus facilitating mating, or in the case of social insects as guides to food
sources. Sesquiterpenes are also secreted as defense substances to fight possible
predators. They have unpleasant odor and sometimes they are toxic. Others are known
as juvenile hormone or they have growth inhibitory or growth-regulatory activity
(Mesmin et al., 2000).f ~ •\ < '
24
The diterpenes represent a large group of terpenoids with a wide range of biological
activities, isolated from a variety of organisms. One of the simplest and most important
acyclic diterpenes is phytol, a reduced form of geranylgeraniol. This terpenoid is
perhaps the most studied of those found in aquatic environments, and it is a side chain
of chlorophyll. Phytol isolated from Lucas volkensii exhibits significant ant tuberculosis
activity (Zhang et al.,2005).
2 .4 .6 T r i t e r p e n e s C 3 0
Triterpenes are not formed by an extension of IPP instead two molecules of famesyl PP
are joined tail to tail to yield the hydrocarbon Squalene (figure 2.5). Squalene is a
precursor for triterpenes and steroids. Several seed oils are quite rich sources of
squalene, e.g. Amaranthus cruentus (Dewick, 2002).
2.4.5. Diterpenes C20
S c h e m e 2 .3 S q u a le n e
2 .4 .7 T e t r a t e r p e n e s C 4 0
The tetraterpenes are represented by only one group of compounds, carotenoids. These
compounds play a role in photosynthesis but are also found in non-photosynthetic plant
tissues, in fungi and bacteria. Formation of a tdtraterpene involves coupling of
25
geranylgeranyl diphosphate (GGPP) in a sequence similar to that of squalene and
triterpenes (Dewick, 2002).
)|/
26
C H A P T E R T H R E E
METHODOLOGY
3.1 E th n o B o t a n ic a l S u r v e y o f C. articulatus L f r o m T h a r a k a M e r u
Ethno botanical studies involve field explorations of indigenous medicinal knowledge
and biodiversity. An ethno botanical survey was carried out in Tharaka-Meru district
during the month of October 2010. Tharaka is found in the larger Meru region in the
former Eastern province. During the time of this study the area was experiencing a
drought that had affected most parts of Kenya and Tharaka being a semi-arid area was
as dry as can be seen in figure 3.1 below.
Figure 3.1 Dry vegetation
Position of Tharaka district in the map of Kenya is shown in figure 3.2.
Figure 3.2 Map of Kenya and Tharaka district.
27
A two day reconnaissance survey was carried out in Tharaka Meru where a total of 7
leaders of the Tharaka Traditional Medicine Practitioners (TMPs) from Ciokariga.
Mukothima. Marimanti and Gatunga divisions met with the researchers at the
Marimanti Methodist Guest House. Each of them gave a brief history on their
traditional medicinal practice in general and displayed the plants they use for treatment
of various diseases as shown in figure 3.3. In the afternoon a brief field visit was made
and TMPs explained the various plants found in the field as shown in figure 3.4. After
this C. articulatus L was selected for detailed study.
3.1.1 Reconnaissance survey
Figure 3.3 TMPs explaining the uses of different plants during the afternoon field visit
G. articulatus L (Ndago) was the plant most mentioned as a medicinal plant being used\ • '
by most of the TMPs and great concern was raised oii its being an endangered species.
Each of them claimed to have used it. After the meeting it was agreed that a one day
Workshop be conducted at the same place with more TMPS from the four divisions of
28
Tharaka (Marimanti, Ciokariga, Mukothima and Gatunga) who practice traditional
medicine and use Cyperus articulatus L. It was also agreed that a man (Mutonga) from
neighboring Mitunguu (Iment South be invited) as he was the one who supplies the
Tharaka people with C. articulatus L from Iment South (neighboring district) during
the dry season when it was scarce in Tharaka. The TMPs were asked to bring C.
articulatus L samples during the workshop.
3 .1 .2 E t h n o - B o t a n ic a l S u r v e y W o r k s h o p
The workshop was conducted by five scientists (one student and four University of
Nairobi lecturers). A semi-structured questionnaire was administered to the registered
traditional herbal medicine practitioners (TMP). Procedures involved in the making of
the medicinal preparation from C. articulatus L were sought. These included procedures
used in the treatment of stomachache, abdominal pain, menstrual period pains, typhoid,
fever, malaria, common cold, cough, flu, running nose, swollen breasts, l?aby skin rush,
blisters, fungal infection, wound. Other ethno medicinal uses that were studied
included; mosquito repellency, mouth freshener and perfume. The sample
questionnaire that was used is shown in Appendix 1. Data was sought regarding the
availability of C. articulatus L , methods of preparation and formulation, dosage,
treatment and outcomes, recurrence as well as patient satisfaction. Data obtained from
the questionnaires was analyzed using the appropriate methods (descriptive statistics)
and the plant collected for taxonomic identification. . ,t ' •\ I '
29
3.2 Plant Material Collection
Fresh root tubers (rhizomes) of C. articulatus L were collected from Ciokariga,
Marimanti, Gatunga. Mukothima and Mitunguu in October 2010 and were identified at
the School of Biological Sciences, University of Nairobi Herbarium. Due to the
ongoing drought during the time of study the plant material collection covered a vast
region and the plant collection exercise took longer than anticipated. As can be seen
from the Figure 3.4 below the plants were dry and tilling the hard ground was not easy.
More field assistants were to be hired and strict supervision done to ensure the right
plants were being harvested.
Figure 3.4 TMPs digging up tubers of C.articulatus L
3 .3 P la n t E x t r a c t io n
The root tubers of C. articulatus L were washed dried under shade and ground using a
Wiley mill from the school of Biological Sciences^Umversity of Nairobi. The powder\ ••\ t
was weighed and yielded 6 kg and 2 kg were preserved in a refrigerator for further
30
analysis. The remaining 4 kg were subjected to serial extraction using dichloromethane
(CH2CI2) ,1:1 dichloromethane/methanol (CH2CI2/ CH3OH) and finally 5% water in
methanol. Each extract was concentrated using a rotatory evaporator at temperatures of
40°C to 60°C with an aspirator vacuum. The 5% water /methanol extract after
concentration was freeze dried at the School of Biological Science University of
Nairobi for 24 hours to ensure the water was removed. The dry powder was tightly
corked and kept in the fridge awaiting further analysis. The crude extracts for each of
the others were combined, tightly corked and stored in the refrigerator to wait for
further processing. The quantities yielded were 100% dichloromethane extract 100
grams, 1:1 methanol/dichloromethane 60 grams, and 5% water in methanol yielded 36
grams. The three extracts were brown in color, oily and all with a pleasant odour.
3 .4 L a b o r a t o r y A n a ly s is
3 .4 .1 G e n e r a l
Merck Silica gel 60 (0.063 -0.200 mm) was used for column chromatography (CC) as
the stationery phase.; PTLC on Merck Silica gel 60 PF254+366, coated on glass plates (20
by 20 cm) to make 1.0 mm layers; Analytical TLC was carried out using aluminum base
plates (0.25 mm) coated with silica gel (6OF254, Merck) and spots visualized under UV
lamp 254-366 nm, followed by spraying with 1% vanillin in H2SO4 spray reagent. EI-
MS spectra were recorded on Agilent Gas chromatography/ mass spectrometer ( GC-
MS).
f ' *\ 1 *
31
Fifty grams of the DCM extract was subjected to column chromatography on silica gel
using varying ratios of ethyl acetate / hexane. A total of 108 fractions were collected
and analyzed using analytical thin layer chromatography (TLC) and the ones found
similar combined. Further purification was carried out using column chromatography
on silica gel and preparative thin layer chromatography (PTLC). After combining they
were renamed A-P. The separation of compounds was monitored using analytical thin
layer chromatography using aluminium coated factory made plates. Fraction C was
weighed and gave three grams and was subjected to further separation as follows. A
column of fifty grams of silica gel was packed with 2% ethyl acetate in hexane. The
three grams were mixed with 5 ml of the 2% ethyl acetate in hexane and charged on the
column. The column was eluted with varying ratios of n-hexane/ethyl acetate (in order
of increasing polarity). A total of forty nine fractions were collected and concentrated
using a rotary evaporator with similar fractions being combined on the, basis of TLC
analysis. Fraction 37 which weighed 100.58 mg was mounted on 6 silica gel precoated
PTLC plates and developed twice in 10% ethyl acetate/hexane. It produced Ci which
when viewed on UV 254-3661™ revealed one sport, these were later sprayed with vanillin
.and also exposed to iodine , the single sports revealed numerous overlapping sports,
that were too difficult to isolate by column chromatography or by preparative thin layer
chromatography (PTLC). Sephadix was also used. The process of isolation of
compounds was rigorous and difficult and did not yiejd any pure compounds. Thesef - 1\ ’ '
methods (CC and PTLC) didn't work for the isolation of these compounds.
3.4.2 Column Chromatography
32
3.4.3 GC-MS
Two microlitres of the crude extract of C.articulatus L 100% DCM was injected
(introduced) in the ion source. This crude extract of C.ariculatus was run on an Agilent
Technologies 7890A GC system connected to an Agilent Technologies 5975C Inert XL
EI/CI MSD mass spectrometer with a triple axes detector. An HP-SMS column with a
length of 30 m and i.d of 0.25 nm was used with a film thickness of 0.25 microns and a
split ratio of 50:1. The oven temperatures were as follows; Starting temperature was at
50 degrees and was held for 3 minutes then ramped up at 10 degrees per minute up to
250 degrees, and held at 250 degrees for 2 minutes. The injection temperature was 250
degrees and the detector temperature was 230 degrees. Helium was used as the carrier
gas. A heated filament that produced a beam of electron (70eV) was used to bombard
the sample. The retention times were as shown in the table in Appendix 2. The diagram
on figure 3.5 shows the GC-MS set up that was used.
Figure 3.5 GC-MS set up f ?[ ^
33
3 .5 I d e n t i f ic a t io n o f th e C o m p o u n d s U s in g G C - M S
Gas chromatography is certainly a very rapid method of separation, since no
preliminary operations are required. It is also a method of choice when only a very
small quantity of oil is available. Constituents of the oil of C. articulatus L were
identified by comparing the experimental gas chromatogram shown in Appendix 2,
retention indices shown in Appendix 3 and MS spectra of the compounds shown in
Appendices 4 to 62 with corresponding reference data (Adams, 1995). For this report
the National Institute of Science and Technology (NIST) library was used. The
percentage compositions of the oils were computed in each case from GC peak areas
shown in Appendix 3. The components of the oils were identified by matching their
retention indices shown in Appendix 2 and mass spectra indicated in Appendices 4 to
62 with those standards of NIST library mass spectra data base of the GC-MS system
from Surrey University Library in the UK. Appendices 4 to 62 gives the spectra
produced during the fragmentation of the compound and below each spectrum is the
spectrum that was searched from National Institute of Science and Technology library.
These spectra were used to produce the structures of the compounds and it is from them
that the fragmentation patterns of the compounds were proposed.
3 .6 A n t ib a c t e r ia l A c t iv i t y A s s a y s
Bacterial clinical isolates Salmonella typhi, Streptococcus pneumoniae and
Staphylococcus aureus were obtained from the department of Medical Microbiology
School of Medicine, University of Nairobi. The strains Were isolated 3 times on nutrientt '
\ ••\ /agar plates, identified and confirmed using the standard bacteriological methods at the
34
Biotechnology Laboratory, Department of Veterinary Anatomy and Physiology,
University of Nairobi, they were maintained on nutrient agar slopes at 4 °C. The
cultures were spread on nutrient agar plate band incubated aerobically at 37 °C
overnight (18-24 hrs) before use. Using plate dilution method the cultures were adjusted
to yield 1:10 colony forming units per ml (cfu/ml). The MIC was taken as the lowest
concentration that inhibited the growth of organisms after incubation and the results
were recorded in Table 4.4.
3 .6 .1 D is c I m p r e g n a t io n
A steady air current was used to dry the plant extract for 24 hours. A volume of 0.5 ml
of 20% dimethyl-sulphate (DMSO) was used to reconstitute the extract. Sterile discs
made from Whitman filter paper No. 1 were soaked in 100 pi of the extract to an
approximate concentration of 20 mg /ml per disc. Discs soaked in 20% dimethyl
sulphate (DMSO) and sterile saline acted as control. The impregnated discs were
sterilized under ultra violet rays (UV) for lhour.
3 .6 .2 S e e d in g o f N u t r ie n t A g a r P la te s
Inoculums of 0.5 ml from each bacteria species were introduced onto the dry, sterile
surface of the nutrient agar (Muller Hinton agar 90XOID) . The seeding rate was
1.0*10'' cfu/ml and a sterile glass spreader was used to make an evenly distributed
culture. The plates were left to dry for 2 hours.
3 .6 .3 B a c t e r ia l S u s c e p t ib i l i t y T e s t in g
Sterile impregnated discs of known concentrations were, carefully placed on the labeled
\seeded plates in duplicates. The plates were allowed to stand for 1 hr to allow diffusion
to take place, and then they were incubated aerobically at 37 °C and examined for zones35
of inhibition after 24 hrs. Discs impregnated with 20% dimethyl sulphate (DMSO) and
normal saline were examined as control. The experiment was repeated 5 times. The
crude extract of 100% CH2CI2 was screened for antibacterial activities against three
different strains of bacteria and the plates used were as shown in the results section. The
inhibition diameters were measured after 24 hours of introducing the extract to the
colonies in the Petri dishes. Different inhibition diameters were recorded for each of the
three experiments and an average reading was calculated and recorded on Table 4.3.
3 .6 .4 M in im u m I n h ib i t io n C o n c e n t r a t io n ( M I C )
The minimum inhibition concentration (MIC) was determined by Microtiter Dilution
Method using microtiter plates. Six hundred milligrams (600 mg) of the extract was
dissolved in 1 ml N, N usually dimethylsulphate (DMSO) and made into a final volume
of 3 ml using nutrient broth (200 mg/ ml). Serial dilution of the 100% CH2CI2 extract
resulted into six concentrations. Racks carrying the dilution test tubes were gently
shaken to mix the content. The negative and positive controls contained only the
nutrient broth. A bacteria suspension containing 1.5 x 106 colony forming units/ml
(cfus) of the test organism was added to each test tube except for the negative control.
The plates were incubated at 37 °C for 24 hours and observed for turbidity. The lowest
concentration of each extract that showed no sign of turbidity indicating growth
inhibition was recorded as the minimum inhibition concentration in mg/ml in Table 4.4.
3 .7 M o s q u it o R e p e lle n t T e s t
This test was carried out using laboratory reared Aedes egyptii mosquitoes. The test was
carried out in a dark room at 25 °C using human baits. Aedes egyptii adult mosquitoes
36
were raised in netted cages at 25 -30 °C from a larval colony. The mosquitoes were fed
for five days on exposed skin of live rabbit ears. One hundred of these adult mosquitoes
were starved for 48 hours and placed in a cage. Human bait was used for the evaluation.
Before each test, the readiness of the mosquitoes to bite was confirmed by having
subject insert an untreated forearm into the test cage. Once subject observed five
mosquito landings on the untreated arm, the arm was withdrawn from the cage and
Cyperus articulatus L crude extract applied as the repellent being tested (Faradin et al.,
2002). The C. articulatus L extract was applied thinly using cotton wool on the bare
forearm from elbow to the fingers and placed in the mosquito cage. Exposure time was
5 min, 15min, 30min and 60min .This experiment was carried out in a semi darkened
room. The number of bites and landings were counted for each exposure period and
recorded in Table 4.5.
3 .8 S t r u c t u r e E lu c id a t io n .
The structures of the compounds were determined using spectroscopic methods. The
chromatogram from the GC and spectra obtained from GC-MS were as shown in
Appendices 4 to 59. These spectra were compared with those of the National institute of
science and technology (NIST) library of Surrey University in the UK and from these
the structures shown on table 4.2 were proposed. The retention times were as shown
in the table in Appendix 2.
t\1 /
37
CHAPTER FOUR
RESULTS AND DISCUSIONS
4 .1 E th n o B o t a n ic a l S u r v e y in T h a r a k a
The information from the questionnaire were analyzed and the following were the
findings. From the information gathered from the TMPs it was clear that they were
using C. articulatus L for treatment of diseases recorded in Table 4.1. Judging from the
responses in the questionnaires and from the little amount of C.articulatus L the
herbalists brought on that day it was clear that C. articulatus L was becoming scarce in
Tharaka. This is because many people from the neighboring districts which had better
growing conditions (loamy soil and shady wet conditions) for the plant had uprooted it.
Due to scarcity of C.articulatus L some people had started using it sparingly. They
understood all its uses and most of what they were mentioning is supported by literature
from other parts of the world, Peru, Brazil, S. Africa, Cameroon and Nigeria. The plant
is becoming scarce in Tharaka and very hard to find at times especially during the dry
season because Tharaka is a dry semi arid area. From the information gathered from the
questionnaires most people agreed that it was becoming less and less with time
4 .2 E t h n o - M e d ic in a l U s e s a n d p r e p a r a t io n m e t h o d s f o r c o n c o c t io n s o f C.
articulatus L f r o m T h a r a k a
The Tharaka people use C. articulatus L in several ways. A summary of the uses
mentioned by the traditional medicine practitioners during the workshop in October
2010 were summarized in the Table 4.1. - • -»
38
Table 4.1 Uses of C.articulatus L from Tharaka Meru
D is e a s e s t r e a t e d /u s e M e t h o d o f p r e p a r a t io n / d o s a g e /u s a g e
Cough Tubers washed chewed 2 tubers 3 times /dayBlocked nose Washed ground powder applied around the nose 3 times /day
Common coldWashed ground and applied around the nose, or taken 1 spoonful 3 times/day
Running nose Tubers cut into pieces boiled taken 1 cup 3times/day
FluTubers washed and chewed or into powder applied around the nose
Fever Washed ground mixed with water and taken 1 cup 3times/day
MalariaWashed ground into powder taken 1 tablespoon 3 times /day or tubers cut into pieces boiled in water taken 1 cup 3 times/day
Pneumonia Whole plant cut into pieces boiled, taken 1 cup 3-times a day.Rheumatism Tubers washed boiled and taken 1 cup 3times/day
WoundsTubers washed ground into powder applied on wounds twice /day
Blisters Washed ground into powder applied on Blisters
Fungal infectionWashed ground into powder, applied on affected Areas (between toes)
Baby skin rushTubers washed ground into powder applied as baby powder or all over the body (affected area of the skin)
TyphoidTubers washed ground mixed with water taken 1 cup 3 times/day
Swollen breastsTubers washed ground into powder applied on swollen breasts twice/day
Stomachache Tubers washed and chewed 2-3 tubers 3 times a dayAbdominal pains Tubers washed and chewed 2-3 tubers 3times/dayMenstrual period pains
Tubers washed and chewed2 tubers 3 times/day
Goat coughWhole plant cut into pieces mixed with water, boiled and given to goats \ ’ '
Mosquito repellency Tubers ground into pounder mixed with water and sprinkled
39
around the houses and on the floor shortly before dusk.
PerfumePowder ground and applied around the armpits, neck and around the waist, sometimes in the olden days mixed with chalk or red earth and applied on the hair.
Mouth freshener Tubers washed peeled and chewed
4 .3 . C h a r a c t e r iz a t io n o f e s s e n t ia l o i ls o f C.articulatus L
4 .3 .1 C o lu m n C h r o m a t o g r a p h y
The TLC of the crude extract of 100%CH2Cl2 revealed UV 254 - 366nm active spots.
Chromatographic separation of the crude extract did not yield any pure compounds,
however characterization using GC-MS resulted to terpenoids with a large number of
mono terpenes (twenty) which are generally known to be difficult to isolate as pure
compounds as mentioned in the literature review. The student leant that Column
chromatography does not always work.
4 .3 .2 .1 C h e m ic a l A n a ly s is o f th e C o m p o u n d s fr o m C. articulatus L f r o m T h a r a k a
GC chromatogram shown in appendix 2 was first produced. And from each of the peaks
shown in the GC chromatogram the spectra for each compound were produced. The
retention indices and the spectra shown in the appendices section were used to identify
the structures of the compounds. From the 100% dichloro methane extract of C.
articulatus L from Tharaka fifty nine compounds were detected. Terpenes accounted for
the highest number of compounds analyzed with forty eight in number (81.36 %). From
the peak areas of these 59 compounds the quantities of each compound was computed.
There were twenty seven sesquiterpenes (45.76%), twenty monoterpenes (33.90%) one
triterpene (1.69%) and eleven other compounds (18.64%). The m^st abundant terpene
was found to be the sesquiterpene a-cubenene. Taffle 4.2 gives the'. GC order number,
compound name, molecular formula, structure, retention time, and relative peak areas of
40
the compounds. The relative peak area percentages were used to compute the quantities
for the compounds. As in the essential oils analyzed by Nureni el al 2006 in Nigeria the
essential oil from Tharaka had terpenes as the major constituents accounting for a total
of 48 out of the 59 compounds. Both the crude extracts and all the fractions from C.
articulatus L. were sweet smelling. This was attributed to the high number of terpenes
in the essential oils. Table 4.2 gives a summary of comparative analyses of these
compounds. The fragmentation pattern in this table compared to those in the NIST
library and suggested these structures. It is from the details in this table and the spectra
that the fragmentation pattern of compound thirty three was proposed by the author.
Table 4.2 Retention Time, M olecular Formula and Corresponding Peak Area % of M aximum for com pounds from C.
articulatus L from Tharaka
G C -
O r d e r
N o .
C o m p o u n d N a m eM o le c u la r
f o r m u laM o le c u la r S t r u c t u r e
R e te n t io n
T im e
R e la t iv e p e a k a r e a % o f m a x im u m
1 IR-a pinene C ioH 16 J 6.16 1.16
2 lS-a pinene C , oH , 6(
6.59 0.57
3 7 vinyl-Bicyclo[4.2.0]oct-l-ene C , oH 14 6.57 2.90
OH
4 Bicyclo[3.1.0] hex-3-ene-2- ol,2methyl 1(1 methyl ethyl),(5,alpha) C , 0H , 4O £ 6.71 1.55
• J
42
r Beta pinene C , oH 16 7.01 3.99
6 alpha phellandrene C , oH ,5
J )7.53 0.66
7 D-Limonene C , oH 15 9.98 1.39
* _8 Eucalyptol C , oH , 80 [
b 8.03 0.52
9
Benzene, 1 -methyl-4( 1 - methylethyl)(cymol, paracymene, P cymene).
C , oH , 4
> - o -7.90 1.39
10 3cyclopentene-1 -acetaldehyde 2,2,3' trimethyl, (a campol). C , oH 160
b
7.68 1.11
43
11Bicyclo[3.1.0]hexane-3- ol,4methylene,-1 -(1 methyl) 1 s-( 1 alpha 3 beta ,5alpha).(sabinol).
C,oH I60 - > 1 9.92 18.25
1 2
Bicylo(3.1.1 )hept-3-en-2-ol4,6,6 trimethy (IS -(alph,.2beta,5alpha). ( (s) cis verbenol)
C,oH 140 10.01 13.13
13Bicyclo[3.2.0]-3- ol,2methylene,6,6,dimethyl C,oH 160
H
_ W -H O----(
n 10.18 0.78
14 Alpha cubenene C15H 24
Lrv13.50 77.89
15 ' -* J
8 Isopropyl-1,5-dimethyl cyclodica - 1,5-diene
\
C15H 24 13.68 2.54
44
r
/ 3H-3a,7methano2.4,5,6,7,8 hexahydro-1,4,9,9tetramethyl-(3a- alpha,4beta,7alpha (cyperana)
C15H24 13.85 77.89
17 Naphthalene 1,2,3,4 tetrahydro-1,1,6 trimethyl (lonene) C,3H,8 13.89 100
18 Caryophylene c 15h 24
V
14.09 5.75
19._
Bicyclo[5.2.0]nonane,2methylene4,8, 8 trimethyl,4,vinyl
c 15h 24 14.08 1.87
20• J
Azulenel,2,3,4,5,6,7,8,octahydro-l- 4-dimethyl-7-[ 1 alpha,7,alpha] (Quaiene)
C|5H24
n
14.30 6.90
45
L I 6-isopropyl-4.8a-dimethy8al-1,2,3,5,6,7,8 , octahydro naphthalene- 2-0
c ,5h 22o
HO5 16.49 19.99
27 1H1,5Benzodiazepine2,3,4,5tetrahydr o-2-methyl C,oH14N2
xz
zx
16.56 14.99
281,2,3,4,5,6 hexahydro 1,1,5,5,tetramethyl-2,4a- methanonaphthalene-7(4a,H)-one
c 15h 220 £ 16.65 24.34
29 Isoaromandrene epoxide c 15h 24o
$
16.74 6.64
30 lH-cycloprop[e]azulene,decahydro- 1,1,7trimethyl-4-methylene c ,5h24 c
9
16.99 12.34
31 2(10)-pinen-3-one CioHi40 c ?V° 10,31 6.77
32 Bicyclo(2.2.1 )heptan-3- one6,6dimethyl-2-methylene C iqHi40 i r 10.31 6.77
47
1442H-CycIoprop[aJnaphthalene-2-one ,1,1 a,4 ,5,6, 7, 7a, 7b octahydro- l,l,7,7a,tetramethyl (la,alpha, 7 alpha,7a, alpha,7b,alpha).(aristolene)
C15H22O\
cr
/
17.61 22.70
45 Caryophylene(l 1) C15H244
18.11 36.31
46 . Alloaromandrene oxide-(l) C,5H220< ?
18.51 37.67
47 Culmorine C15H24O2
/H O
o h \
/
18.95 32.18
._>•- v
48
* J
Ciz-2-alpha Bisobolene epoxide C15H24Oy
19.03 42.16
50
I
49 5-Isopropenyl-2-methyl-7- oxabicyclo[4.1,0]heptan-2-ol C,0Hl5O2
O H
> 19.23 38.44
50
Tricyclo[4.3.0.0](7,9)nonane2,2,5,5,8,8hexamethyl-alpha.6beta,7alpha,9alpha.2,2,5,5,8,8-Hexamethyl-tricyclo[4.3.0.0*7,9*]nonane
C15H26X
X
x ;19.39 22.65
51 Longifolenaldehide C,5H240e
X 19.68 36.72
52
------ 1
1H-Inden-1 ol-2,4,5,6,7,7a hexahydro-4,4,7 a-trimethyl c ,2h 20o
O H
20.76 8.95
51
53
53). 1 H-Cycloprop[e]azulene,1 a,2,3,5,6,7,7a,7b,octahydro,-1,1,4,7,tetramethyl,9,1 aR( 1 a,alpha,7a,beta,7,alpha
C15H24 23.78 3.58
54 54). Methyl4,6-decadienyl ether C„HIgO (CH3)2-0-(CH)5(CH)4 25.33 8.9655 55). Dodecanoic acid, tetradecyl ester C26H52O2 (CH3)2(CH2)23CHO-0 26.80 7.7656 56). Dodecanoic acid, hexadecyl ester C28H58O2 (CH3)2(CH2)26COO 28.21 18.15
57 57). Succinic acid, heptyl tridec-2-ynl ester
qsccnu CH3)2(CH2),5CH2)2COCH
o c o o 28.92 2.06
58 58). Dodecanoic acid ,octadecyl ester C31H62O2 CH3)2(CH2)28COO 29.76 4.02
59 59). Longipinocarveol,trans c 15h220 28.62 1.27
52
4 .3 .2 .2 P r o p o s e d F r a g m e n t a t io n P a t t e r n f o r C o m p o u n d 3 3
Compound 36 was chosen to illustrate the fragmentation pattern for terpenes. The spectrum in
figure 4.1 was used. It is from this spectrum that the fragmentation pattern for compound 33 was
proposed by the author. Compound 33 contains the hetero atom oxygen which has two lone pairs
of electrons. An electron was knocked from the lone pair of electrons by the beam of electrons
that bombarded the sample at the ionization chamber. This created the molecular ion shown in
the first step in Scheme 4.1 with a mass to charge ratio (m/z) of 152. The bond adjacent to the
hetero atom formed a triple bond with oxygen at C-l. The remaining electron forms a radical at
C-6. Through a hydride shift the radical shifted to C-2 and using this radical the bond adjacent to
C-3 broke to form a double bond between C-2 and C-3. The radical shifted to C-4. This coupled
with an electron from the bond adjacent to C-6 and cleaved the bond. This gave rise to an
ethylene and another positive ion (6-methylheptan-l ol) with an m/z 123. Carbon monoxide was
then lost to form another positive ion with m/z 95. Then an ethylyne was lost to produce another/
positive ion of m/z 69 and eventually loss of three consecutive methyls to produce positive
fragments of m/z 55, m/z 41 and m/z 27 consecutively. This fragmentation pattern is illustrated
stepwise in Scheme 4.1.
53
S ch e m e 4 .1 : P r o p o s e d F r a g m e n t a t io n P a t t e r n f o r C o m p o u n d 3 3
>\1 /
54
library Searched
qualityID
C:\Database\NIST08.L94lH-Cycloprop[e]azulene, decahydro-1,1,7-trimethyl-4-methylene-, [laR- (la.alpha.,4a.alpha.,7.alpha.,7a.beta.,7b.alpha.)]-
Figure 4.1 mass spectrum for compound 33
4.4 In-vitro A n t im ic r o b ia l A c t iv it ie s
The extract obtained using 100% CH2CI2 exhibited activity against all bactaria strains tested
{Staphylococcus aureus, Streptococcus pneumonia and Salmonella typhi). This is as shown in
plates 4.1 and 4.2 below. The inhibition zones for the strains tested were : S.aureus 12.0 mm.
S.pneumonia 9.0 mm and S. typhi 8.5 mm.
t\
55
P la te 4 .1 Staphylococcus aureus
P late 4 .2 Streptococcus pneumonia
T a b le 4 .3 A n t i m ic r o b i a l a c t iv i t i e s o f t h e l 0 0 % C H 2C I2 c r u d e e x t r a c t o f C.articulatus L f r o m
T h a r a k a .
Bacteria strain Staphylococcus aureus StreptococcusPneumonia
SalmonellaTyphi
.Average inhibition diameter_Neat concentration 15 mm 12 mm 1 mmJj5-concentration 12 mm 8.5 mm 9 mmJjlO- concentration 9 mm 8 mm 8 mmJilpO-concentration 8 mm 9 mm 7 mm
t\ /
56
4,4.1 M in im u m I n h ib i t io n C o n c e n t r a t io n
The minimum inhibition concentrations were taken and recorded. At 100 % concentration
(undiluted crude extract of dichloro methane) there was no growth observed for the three
bacterial strains. At lOmg/ml concentration there was no growth for Staphylococcus aureus but
there was partial growth for both S. pneumonia and S. typhi. At 1 mg/ml concentration there was
partial growth for Staphylococcus aureus and full growth for Streptococcus pneumonia and
Salmonella typhi. At 0.1 mg/ml concentration there was full growth for all the three bacteria
strains.
4.5 Mosquito Repellent Test Results
When the untreated arm was placed in the cage five landings were observed indicating the
readiness of the mosquitoes to bite. When the 100% CH2CI2 extract was smeared on the forearm
of the experimenter and the hand immediately placed inside a darkened cage with approximately
100 adult mosquitoes that had been starved for 48 hours, the following was observed;
1. The mosquitoes were ready to bite as observed in figure 4.5a, five landings were recorded.
2. When the hand with extract was introduced into the cage all the mosquitoes flew away and not
a single landing was observed.
3. When the untreated arm was re-introduced into the cage the mosquitoes landed and baits were
recorded in Table 4.3 below.
T a b le 4 .4 N u m b e r o f b i t e s a t d i f f e r e n t p e r i o d s o f e x p o s u r e
Period of exposureControl Hand/bites LandingsTreated Hand/bites Undim
5minutes 15minutes 30minutes 60minutes30 42 52 5539 46 61 630 0 0 \ 0 'i '1 0 0 0 ' •
57
The percentage bites were calculated by the formula; - x 100 , Where B = number of bites andA
X —number of landings.
0
t\
I /
58
C H A P T E R f i v e
5.0 CONCLUSIONS AND RECOMMENDATIONS
5.1 C o n c lu s io n s
The traditional medicine practitioners in Tharaka were using C. articulatus L for treatment of
various diseases. Form the ethno-botanical survey it was evident that most people had uprooted
C. articulatus L from their farms. C. articulatus L was becoming scarce as was evidenced by the
long distance herbalists covered searching for the plant. The constituents of C. articulatus L from
Tharaka had the sesquiterpene a- cubenene as the major component. The compound a- cubenene
was not detected in all the other essential oils extracted from C. articulatus L from Nigeria, S.
Africa, Brazil, Japan, Taiwan, Thailand, Hawaii and Philippines. The following compounds were
found in both the essential oils of C. articulatus L from Tharaka and the one reported from
Nigeria by Nureni et al 2006; Caryophyllene, Caryophyllene oxide, cymene, pinene, gurjunene,
and muurolene. The following compounds were detected from the essential oils of C. articulatus
L from Tharaka and absent in that from Nigeria; phellandrene, D-limonene, eucalyptol,
mertenol, thymol, alloaromandrene oxide, culmarine, lsoaromandrene epoxide and a-cubenene.
Cyperene was a compound reported from C. articulatus grown in Brazil, Japan, Taiwan,
Thailand, Hawaii, Philippines, and C. rotundus from S. Africa but was missing in C. articulatus
L from Tharaka in Kenya. C. articulatus L from Nigeria was reported by Nureni et al 2006 to
have cyperotundone as its major component and this was absent in the C. articulatus L from
Tharaka. Comparing the results of C. articulatus L from Tharaka with those previously reported
in literature on essential oils of C. articulatus L and other related species it is apparent that there
are many differences regarding the major constituents of the oil and its other components. This
could be due to different climatic and environmental conditions in Kenya and other parts of the
world as explained by Oladipupo et al., 2009. This means that the Kenyan essential oil is unique.
As in the previous findings the sesquiterpenes were the major components, twenty seven in
number and going by the computation of relative peak area percentages given in table 4.2 they
were in larger quantities than the monoterpenes which were twenty in number and in smaller
quantities. This was the case in the Nigerian essential oiLwhere the sesquiterpenes were major
and the monoterpenes were the minor components. The number of compounds indentified in
59
Kenya was greater than that in Nigeria. Kenyan essential oil had 59 compounds, Nigerian- red
tubers-37 and black tubers-47. This is could be because in Kenya the method used was solvent
extraction which is known to extract more compounds than all the other methods, in Nigeria
hydro-distillation was the method extraction used. Attempts to isolate the essential oils of C.
articulatus L obtained by using 100 % CH2CI2 extract from Tharaka were unsuccessful. This was
attributed to the presence of a large number of number of monoterpenes which have very close
retention values and therefore difficult to separate by column chromatography. Crystallization
also failed as a method of compounds purification in this study because sesquiterpenes are oils at
room temperature and do not usually crystallize as stated by Mesmin et al., 2001. C. articulatus
l from Tharaka had a lot of similarities with that extracted from Brazil and other parts of the
world, this means that it has the potential for drugs and other products ranging from mosquito
repellent, perfumes, air freshener, mouth-freshener and a range of other cosmetic products as
evidenced in the results in chapter four of this report. The crude extract of C. articulatus L was
active against the three bacterial strains: S. pneumonia, S. aureus and S. typhi. The best and most
effective dose was the undiluted 100% CH2CI2 extract for all the bacterial strains except for S.
aureus that was inhibited in both undiluted 100% CH2CI2 extract and the 1 mg/ml according to
the records on tables 4.3 and 4.4. The extract was repellent against the mosquito A. egyptii with a
repellency of 100% as recorded on table 4.5 which supported the TMPs claim that it was being
used as an insect repellent. The use of C. articulatus L by the entire Meru community for
treatment of typhoid, stomachache, blisters and wounds could be related to its anti-bacterial
activities reported in this study. It is being reported for the first time here that C. articulatus L
from Tharaka has activity against S. typhi, S.aureus and S. pneumoniae. We also report for the
first time that the crude extract of C. articulatus L from Tharaka has repellent activity against the
mosquito Aedes egyptii.
S-2 R ecom m endations
The farmers in Meru should be encouraged to plant more C. articulatus L as its oil composition
ls similar to that of France, Brazil and other countries that are using it in cosmetics and(
Perfumery industry in hair care, fragrance, oral care, sjcin care, and deodorant. We here
rec°mmend that formulation as a perfume, deodorant, air freshener, mouth-freshener and a60
niosquito repellent be done on this plant. It should also be examined for anti- allergenic effects
because its sweet smell does not seem to affect people who are allergic to other commercial
perfumes. Use of botanical derivatives in mosquito control instead of synthetic insecticides will
reduce the cost and risks of environmental pollution. Further studies on identification of active
compounds, toxicity and field trials are needed to recommend the active fractions of this plant
extracts for development of eco-friendly chemicals and indigenous plant base oil for protection
against the bites of insects and the above mentioned uses. Other chromatographic methods
example preparative gas chromatography (GC) should be tried for isolating the pure compounds
and further tests be done on the pure compounds. This is because sesquiterpenes structures may
serve as new prototypes, or templates for synthetic organic chemists to use in the design of
potentially superior chemotherapeutic or otherwise biologically active agents. The research
should also be extended to the other Cyperus species found in the area of study like Cyperus
rotundus which from S. Africa is reported to have related compounds.
61
References
iamSi R .P (1995). Identification of essential oils compounds by Gas Chromatography/Mass
spectrometry. Carol Stream, IL, USA: Allured Publishing Corporation.
1 chan P . R . , B. Nageshwar R., Mamatha B. , Satish R. B.S. (2009). Thymol, a naturally occurring
monocyclic dietary phenolic compound protects Chinese hamster lung fibroblasts from radiation-
induced cytotoxicity. Mutation Research. 6 8 0 . 70-77.
Lnhard T., Wolfgang B ., Mario W. (2011). Identification of Thymol phase
Metabolites in human urine by headspace sorptive extraction combined with thermal
desorption and gas chromatography. Journal of Pharmaceutical and Biomedical Analysis
I mass spectrometry. 56.64-69.
fickell C. ( 2003). RMS A-Z Encyclopedia of garden plants. 3rd Edition. Dorling
Kindersley, London ISBN0-7513-3738-2.
lani.M., Menghini F., Pagiotti R., Menghini A., and Fatichenti F. (2000). Antimicrobial I /W
Properties of essential oil of Satureja montana L. on pathogenic and spoilage yeast.
Biotechnology Letters. 22. 1007-1010.
-G, Baruah I., Talukdar P .K and Das S. C (2003). Evaluation of botanicals as repellents
against mosquitoes. Journal of vector borne diseases. 40.49-53.
H'«kp. M (2002). Medicinal Natural Products -A biosynthetic Approach. 2nd Edition. John Wiley &
sons Ltd.
*n" ita D . D . , Parimala G., Saravana D. S., Tapan C. (2011). Effects of
Thymol on peripheral blood mononuclear cell PBMand aciite peripheral blood
m°nonuclear cell line HL-160. Chemco-Biological interactions. 193.97-106.62
.Jip o.T ( 2006). Indigenous knowledge in nature conservation and utilization. Report on nature \Ar'
conservation and natural disaster management-role of indigenous knowledge in Swaziland.
UNEP and University of Swaziland, Mbabane.pp 21-51.
L Habershv, Mansour R.M.A, .Zahran M.A, El-Hadidi M .N, Saleh (1989). Leaf flavanoids of
Cyperus in Egypt.Biochemical systematic and Ecology. 17(3). 191-195.
R u k a n g ir a E . (2001). The African herbal industry; constraints and challenges. The
natural Products and Cosmeceutcals 2001 conference. Erboristic Domani.
Faradin M . S, John F.D. (2002).Comparative efficacy of repellents against mosquito
bites. New England Journal of medicine. 347.13-18.
Mesmin M . S., Wilfried A. K. (.2001).Chemical sturdy of the essential oil of
Cyperus rotundus. Phytochemistry. 58.799-810.
Farnsworth. N. R., Soejarto D. D. (1985). Potential consequences of plant extinction in the
United States on the current and future availability of prescription drugs. Econ. Bot.
39(3).231-240./
eoffrey D. B. (1996). The Biosynthesis of Artemisinin (Qinghaosu) and the
Phytochemistry of Artemisia annua L. (Qinghao). Molecules. 15. 7603-7698.
“rdon-G. K.D., Ward C.J.. Edwards J .J. (2006). Studies in Cyperaceae in Southern Africa.
Cyperus articulatus /..and Cyperus corymbosus Rottb.South African Journal of
Botany. 72.147-149.
^BaM. C.F., Antonio J.C. S., Beatriz S. M. T. and Graziela G. B. (2006).
Total synthensis of the sesquiterpenes beta corymbol and Corymbolone. 62. 9232-9236.■
*’ ^°umaya, Abdelwahed, Afef, Ammar, Ribai B., Hayder, Nawel (2005).,Chemicalp
Ornposition. Antibacterial and Antimutagenic Activities of Essential Oil from (Tunisian)63
Cyperus rotundus
Inborn AD (1999). The discovery of drugs from higher plants. Biotechnology.
26.108.
ucinweton S.M. .Roserio f., Patricia F. L., Marcos L. C., Lulio C.- Filho, M.Angela A. Meireles
(2008). Phase equiribrium measurements for C02 pripioca
extract at high pressure. Journal of Supercritical Fluids. 48.126-130.
[hang L., Demain A. L. (2005) Natural Products: Drug Discovery and Therapeutic Medicine
Humana Press Inc., Totowa, NJ.
laria D., Zoghbi G.B., Eloisa H.A., Andrade, Lea M.M., Carreira E., Rocha A.S. (2008).
Comparison of main components of the essential oils of “Priprioca: Cyperus articulatus,
VarL. Articulatus L.C.articulatus var. nodosus L.C. prolixus Kunth and Cyperus rotundus
LC Journal of Essential oils Res. 20.42-45.
jlesmin M. S., Wilfried A. K.., Kubeczka K.H. (2000). Isolation and structure
elucidation of essential oil constituents.-P.H.D dissertation University of Hamburg, faculty
of Chemistry- Cameroon.
tamin M. S., Wilfried A. K. (.2001).Chemical study of the essential oil of Cyperus
rotundus. Phytochemistry. 58.799-810.
»hl°u m . (2004). Review article on methods to study the Phytochemistry and Bioactivity
ot essential oils. Phytochemistry research. 18.435-448.
“tamniad Z., Mushtaq A., Mir A. K.., Shazia S., Gul J., Farooq A.,
Asma J., Ghulam M. S., Shabnum S., Amin S., Abdul N., .. - nt ■ •
Sarfaraz K. M. (201 l).Chemotaxonomic clarification of pharmaceutically important
sPecies of Cyperus L. African Journal of Pharmacy and Pharmacology. 5(1). 67-75.
„ihaura N. Keriko J. M., Derese S., Yenesew A. Rukunga G.M (2011) Investigation of some
Medicinal plants traditionally used for treatment of Malaria in Kenya as potential sources of
ant Malaria drugs .Experimental parasitology. 127. 609-626.
Ijyingi M. O, James M. M., Adamson L. L., Cyrus G W., Kipsengeret B.
K., Humphrey F K„ Rahab W M. and William O. O. (2008).
Ethnopharmacological survey of Samburu district, Kenya. Journal of Ethnobiology and
ethnomedicine. 4.14.
leville G.A (1968). Indentification of ketones in Cyperus articulatus .NMR and mass spectral
examination of the 2-4 dinitrophenly hydrazones. Tetrahedron. 24.3891.
'igo E.B., Lingenhoehl K„ Rakotonirin A., Olpe H.R, Schmutz M., Rakotonirina S. (2004) .Ions and
amino acids analysis of Cyperus articulatusL.(Cyperaceae) extracts and the effects of the
latter on Oocytes expressing some receptors.Journal of Ethnophamarcology. 95.303-309.
(oE. B., Rakotonirin S., Chumutza. M. M. (2001). Biological and phytohemical screening of
plants. Journal of Ethno-phamacology. 76.145-150.
!° E B., Schmutz M., Meyer C., Bopelet M., Portet C., Jeker A.,.Rakotonirina S.V, H.R. Olpe,
P-Herring. (2001). Anticonversant properties of the methanolic extract of Cyperus articulatus
(Cyperaceae).Journal of Ethnopharmacology. 76.145-150
r ni O.O, Lamidi A. A., Isiaka A. O., Kasali A. A. (2006). Constituents of Rhizomes essential oils
of two types of Cyperus articulatus L. grown \ 1 '
in Nigeria. Journal of Essential oil. 18. 604-606.
65
e u. T., Sondengam R .G., Martin B.L, Bod M.T. (1988). Mandassindione and other
sesquiterpenic ketones from cyperusarticulatus.Phytochemistry. 27.3319-3321.
plandiP0 A.L., Adebola. O. O. (2009); Chemical composition of essential oils of
Cyperus rotundus L. From South Africa.Molecules. 14.2909-2917.
Lintree Nutrition(2006).inc.Carson city, NV8970.
#s i i . f and Recio M.C. (2005). Medicinal plants and antimicrobial activity. Journal of
Ethnopharmacology 100 (1-2). 80-84.
Lgbo R. N., Anuagusi C. L., Amadi J.E. (2009).Advances in selected medicinal and aromatic
Plants indigenous to Africa. Journal of medicinal plants Research. 3(2).86-95.
|kunga G.M, Muregi F.W., Omar S.A., Gathiriwa J.W., Muthaura C.N., Peter M.G.,
Heydenereich M.. Mungai G.M. (2008). Anti plasmodial activity of the extracts of and two
sesquiterpenes from Cyperus articulatus. Fitoterapia. 79.188-190.
ithC. D., Shannon F., Evangelos B., Melony M. and Valerie B.
(2001). Qualitative Analysis of citrus fruit extracts by GC-MS: An undergraduate
Experiment. Chem. Educator. 6.28-31.
«mons T.W .G (1996). Organic Chemistry, 5thEdition, J.Willy& sons.inc.P 1053.
‘"forth P. S. (2006). Natural product chemistry at a glance. Blackwell publishing
Company inc.350 main street, Malden, M.A 02148-5020, USA.
“r L. (2006). Technical data for piripiri (Cyperus articulatus).
'#r JLS, Rabe T, McGaw LJ, Jager AK, van S. J (2001). Towards the scientific validation of
traditional medicinal plants. Plant Growth Regulation. 34. 23-37• ' -v
H 1 S. R., Elisabeth N. B., Alice R., Merc B. (2000). Sedative properties of the decotion of the
fhizome of Cyperus articulatus. Fitoterapia. 72.22-29.
66
jHO (2003). WHO guidelines on good agricultural and collection practices (GACP)
for medicinal plants. World Health Organization, Geneva.
6 ,h I n t e r n a t io n a l Congress on Perfumery and Natural Raw Materials Grasse- France 2007
A P P E N D I C E S
E V A L U A T IO N O F M E D IC IN A L P L A N T P R E P A R A T IO N S U S E D IN T H E T R E A T M E N T O F F E V E R A N D P A INSerial number of the questionnaire..................Name of interviewer.............................. Date.....................P A R T O N E : C O N S E N T A R E S E A C H E R ’S D E C L A R A T IO N
1. The following research will be undertaken with respect to the indigenous knowledge and intellectual proprietary rights of the herbal practitioners.
2. We will at no time initiate or conduct practices that are deemed to obtain information from the respondents by intimidation, coercion or false pretence.
3. We will be under no obligation to edit or tamper with the information provided by the respondents.
4. The information collected will be used for the described research purpose and not any undisclosed intentions.
Researchers:K a r a m b u M u r i i t h i D r . M b a a b u M a t h iu P r o f . J .O M id iw o
P r o f . .S .G .K ia m a D r . J .M W a n j o h i
R E S P O N D E N T S C O N S E N T A G R E E M E N TI ................................................................. hereby agree to participate in the study with my fullconsent and conscience, and declare that to the best of my knowledge the information that 1 have provided is true, accurate and complete.
Appendix 1 Questionnaire used for traditional medicine practitioners
Signature / Thumb print
f\ /
68
P A R T T W O A : B I O D A T A
Name.......................................age(years)............... gender...Location of practice..................... in..................DivisionNumber of years of practice.................................................H o w did you acquire your skills.............................................Level of education*(none, primary, secondary, college, other....................Contact address/telephone..................................................B: M E D I C I N A L U S E O F C Y P E R U S A R T I C U L A T U S
1. Which diseases / conditions do you treat with Cyperus articulatus L (Ndago)
Disease/condition Plant part used
How do you prepare?
How much do you administer/ how many times in a day
Remarks
1234567
i) Is Cyperus articulatus L (Ndago) readily available?............................................ii) How far (long) do you go (take) to get Ndago today compared to It) years ago?iii) Is Ndago cultivated or collected wildly from the forest?
(a) cultivated(b) collected from the forest
iv) Apart from the medicinal uses what else do you use Ndago for?
v) What part(s) of the plant do you use?1. Stem2. Roots3. Tubers4. Flowers
vi) How do you prepare the medicine? ( detail the entire process of harvest, processing and mixing including ratios until ready for taking)
>........................
69
vii) How long can you keep the medicine before it goes bad?
viii) How does the patient take the medicine?ix) How long does the patient take to get well?.............................x) What problems do you encounter in herbal medicine practice?
xi) Is there any reported case of toxicity?......xii) What amount of medicine do you give to:
1. Adults2. Children
xiii) Are there any differences in usage by:1. Male2. Female
t\
-v
70
A p p e n d ix 2.GC-MS Results for 100% DCM Extract
EEPCst'pIIMT
S
pe
12345
6 7 O 910
11 121314 l b
1617181920
2 1 222324 2 b
262 7 282930
3132333435
3637383 9 4 0
Data Path Data File A c q O n O p e r a t o r S a m p l e M l s cA L S v i a l
MethodT it le
: C:\msdchem\l\data\Staff\M I,angat\: K E O . L>: 11 Jan 2011 11:41
: K E O
: l Sample Multiplier: 1
Parameters: autointl.eChemSLation
C :\mndchom\1\methodo\fltandard.M
1010770 248598841162000 2444588610755194 4126980091627781 34256020
42 14 . 368 1965 1972 1977 VV
4 3 14 . 4 4 4 197 7 1985 1989 VV
4 4 14 . 62 9 2 005 2017 2025 W4 5 14 . 706 2025 203 1 2035 W
4 6 14 . 7 93 2035 204 6 206 3 W4 7 15 . 04 6 2077 2090 2093 VV
4 8 15 . 097 2093 2099 2 103 VV4 9 15 . 175 2 103 2113 2 122 VV50 1 5 . 3 14 2 122 2 137 2 14 J PV
51 15 . 377 2 14 3 2 14 8 2 158 W52 1 5 . 4 59 2 158 2 162 2 16 5 VV53 15 . 53 3 2 165 2175 2 18 1 VV5 4 15 . 64 7 2 18 1 2195 2201 VV55 15 . 702 2201 2205 22 10 W
56 15 . 794 2210 222 1 2228 W5 7 15 . 8 94 2228 2238 224 2 W58 15 . 96 9 224 2 2251 2254 W59 16 . 04 7 2254 2265 2269 W60 16 . 080 2269 2271 2274 w
6 1 16 . 120 2274 22 79 2284 w62 16 . 194 2284 2291 2307 w6 3 16 . 3 14 2 3 07 23 12 23 15 VV64 16 . 3 53 2315 2 3 18 2 32 1 VV65 16 . 4 2 7 2 321 2 3 3 1 23 3 5 VV
66 16 . 4 90 2335 2344 2 34 9 VV6 7 16 . 56 1 2 34 9 23 55 2 360 VV68 16 . 64 9 2360 23 70 2 3 83 VV6 9 16 . 74 5 2 3 83 23 87 2 3 91 w7 0 16 . 7 92 2 3 91 23 95 24 01 w
7 1 16 . 84 0 24 01 24 04 2 4 06 VV72 16 . 953 24 06 24 2 3 24 2 7 w73 17 . 002 2 4 27 24 3 2 24 36 w74 17 . 053 24 36 24 4 1 24 4 4 w
75 17 . 095 24 4 4 24 4 8 24 53 w
76 1 7 . 26 3 24 53 2477 24 0 0 w77 1 7 . 325 24 80 24 00 2501 V V70 17 . 4 26 2501 2506 2510 VV79 17 . 503 2510 2 5 1 9 252 2 V V80 17 . 54 9 2522 2528 253 5 w
81 17 . 6 19 2535 2 54 0 254 5 w82 1 7 . 6 84 254 5 2 551 2557 w
8 3 1 7 . 74 4 2557 2 562 2 5 6 5 V V04 1 7 . 705 2565 2569 2573 V V85 1 7 . 0 32 2573 2577 2586 w
86 17 . 950 2586 2598 2603 VV
3 t a r i c 3 a r d . M M o n J u l 18 09:39:: 4 6
^ 7 / 3 X V O > v V
n d a r d . M M o n J u l 1 8 09 : 3 9:46 2 O
7302692 196204741 1904768 47229419 4046256 84307337 8081650 203452896 4773749 136601977
824152
2.00%1 . 9 7 %33.28%2 . 7 6 %
1 5 . 8 2 % 3 . 8 1 % 6 . 8 0 % 16.4 1% 11.02 %
0.106%0.104%1.752%0.145%
0.033%O . 200% 0.350%
8 6 3 % 5 8 0 %
802530
300404417412073768999
11992701212624
3 7 2 8 7 1 O 2 9 6 4 5 3 3
1520255
4 94 2 976
1 1 8 3 8 3 9 9 0 9 . 5 5 % o 5 0 2 %
1 7 2 9 8 1 2 4 1 . 4 0 % O 0 7 3 %
1 1 6 2 2 5 3 3 4 9 . 3 7 % O 4 9 3 %
2 0 0 7 1 0 9 0 1 1 6 . 1 9 % o . 8 5 2 %
2 1 0 8 8 7 4 0 1 . 7 0 % o • 0 9 0 %
8 8 6 1 0 0 3 7 7 . 1 5 % () 3 7 6 %
4 2 6 2 4 0 7 3 3 . 4 4 % 0 1 8 1 %
9 4 8 6 1 3 7 0 7 . 6 5 % () 4 0 3 %
3 8 3 3 3 1 4 1 9 3 0 . 9 2 % 1 . 6 2 7 %
1 4 1 6 1 0 2 2 6 1 1 . 4 2 % o 6 0 1 %
4 8 4 3 7 7 7 4 8 3 9 . 0 6 % 2 . 0 5 6 %
6 4 0 1 5 7 • 3 i 5 1 . 6 3 % 2 . 7 1 7 %
2 3 7 2 8 1 3 2 1 . 9 1 % o 1 0 1 %
2 3 1 2 0 2 6 2 1 . 8 6 % o 0 9 8 %
1 3 6 3 7 3 7 2 0 1 1 . O O % o 5 7 9 %
2 4 7 8 0 3 2 8 7 1 9 . 9 9 % 1 0 5 2 %
1 8 5 8 9 1 1 1 7 1 4 . 9 9 % o . 7 0 9 %
3 0 1 7 9 9 9 0 0 2 4 . 3 4 % 1 2 0 1 %
0 2 2 7 2 0 0 6 6 . 6 4 % o 3 4 9 %
7 5 6 4 2 3 0 4 6 . 1 0 % o 3 2 1 %
2 5 2 0 7 9 9 2 2 . 0 3 % o 1 0 7 %
2 0 2 5 0 4 7 4 7 1 6 . 33% / o 8 5 9 %
1 5 3 0 3 1 4 1 4 1 2 . 3 4 % o 6 4 9 %
1 0 5 6 0 4 7 2 0 8 . 5 2 % o 4 4 8 %
1 5 3 5 6 2 3 6 8 1 2 . 3 8 % o 6 5 2 %
0 7 2 1 9 5 9 4 0 7 0 . 3 4 % 3 . 7 0 2 %
1 0 4 4 3 8 3 5 4 9 8 4 . 2 3 % 4 . 4 3 3 %
2 1 3 2 5 8 4 3 0 1 7 . 2 0 % o 9 0 5 %
4 9 4 3 8 7 8 1 2 3 9 . 8 7 % 2 . 0 9 8 %
6 5 5 5 9 5 5 8 9 5 2 . 8 7 % 2 . 7 8 2 %
2 8 1 4 7 3 6 4 3 2 2 . 7 0 % 1 . 1 9 5 %
2 6 9 4 9 5 1 5 6 2 1 . 7 3 % 1 1 4 4 %
1 4 5 2 1 0 6 5 4 1 1 . 7 1 % o 6 1 6 %
1 5 0 7 9 8 3 3 0 1 2 . 1 6 % o 6 4 0 %
1 7 7 8 2 2 4 9 1 1 4 . 3 4 % o 7 5 5 %
2 5 1 4 5 9 7 9 6 2 O . 2 8 % 1 0 6 7 %
'i fc* . 9 O % O . 3 6 J %
71
Data Path Data File Acq On Operator Sample MiscALS Vial
C :\msdchem\l\data\Staf f\M_Langat\ KEO.D11 Jan 2011 11:41
KEO
1 Sample Multiplier: 1
Integration Parameters: autointl.e Integrator: ChemStation
Method : C:\msdchem\l\methods\Standard.MTitle :
132 25.343 3875 3890 3915 W 5431310 111075986 8.96%133 25.708 3947 3953 3962 W 740704 16683416 1.35%134 26.816 4139 4147 4160 W 4911600 96215719 7.76%135 28.061 4353 4365 4378 BV 4 618437 17466143 1.41%
136 28.206 4378 4390 4409 W 10915950 225095371 18.15%137 28.618 4455 4462 4470 W 6 559191 15736951 1.27%138 28.698 4470 4476 4487 W 4 729356 17709918 1.43%139 28.915 4506 4514 4531 BV 1102900 25531440 2.06%140 29.612 4620 4636 4652 BV 5 605723 25038292 2.02%
141 29.759 4652 4661 4674 W 6 1567452 49810212 4.02%
Sum of corrected areas: 23561624684
t\I
i 'i
0.471%0.071%0.408%0.074%
0.955%0.067%0.075%0.108%0.106%
0 .211%
72
Appendix 3 Gas Chromatogram for thel00% Crude Extract
Data Path Data File Acq On Operator S a m p l e Misc ALS Vial
C :\msdchem\l\data\Staff\M_Langat\ KEO.D11 Jan 2011 11:41
KEO
1 Sample Multiplier: 1
Integration Parameters: autointl.e Integrator: ChemStation
Method : C:\msdchem\l\methods\Standard.MTitle :
Standard.M Mon Jul 18 m i l
73
Appendix 4 Mass Spectrum for Compound 1
Library Searched : C:\Database\NIST08.L Quality : 96
: IS-.alpha.-PineneI D
Abundance
9 0 0 0
800 0
7 0 0 0
6 0 0 0
S 0 0 0 ‘
4 0 0 0
3 0 0 0
2000
1000
S can 537 (6 .1 5 9 min): K E O .D U a ta .m s 93.1
7 7 .0
5 3 1 67.1
O'' i i j i > 11 j‘MVt | m c,,-
1 0 5 1121.1
1 3 6 1
r r i [ * t - j i r r i j i i i r | i i i i [ i t t f j r n2 0 6 1
m /z - > 20 30 4 0 5 0 6 0 7 0 80 90 1 0 0 110 120 130 140 150 1 6 0 1 7 0 180 190 2 0 0 2 1 0abundance (115514 IS-alpha •I’lnni'c
9 3 .0
9 0 0 0
8 0 0 0
700 0
6 0 0 0
500 0
4 0 0 0
300 0
2000
1000
77.0
4 1 .0
27.0 53.0 670 105 .0121.0
1 36 .0
m i I i . i i | i i i ' | i i i i | i i i i | i i ' i | i 1 1 1 1 i i i i | 11 i i | 11 1 1 1 ' 11 i 11 i n 11 i i i | i - m | i i i i | 11 i i | i 1 1 1 [ 11 m | r r i i 11 . . , .
T i/z -> 20 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100 110 120 130 140 1 5 0 160 170 180 190 2 0 0 2 1 0
74
Appendix 5 Mass Spectrum for Compound 2
Library Searched : C:\Database\NIST08.LQuality : 96ID : 1R-.alpha.-Pinene
A bundance S can 537 (6 .1 5 9 min): KEO.OVdata.m s
f\I /
75
Library Searched : C:\Database\NISTOB.L Quality : 64ID : Bicyclo[4.2.0]oct-l-ene, 7-endo-ethenyl-
A b jn d a n c e S ca n 6 0 9 (6.571 m in): KEO .DVdala m s
Appendix 6 Mass Spectrum for Compound 3
• ' *Vl ' '\ »
76
Appendix 7 Mass Spectrum for Compound 4
Library Searched : C:\Database\NIST08.LQuality ID
Abundance
900 0
800 0
700 0
600 0
500 0
400 0
3000
2000
1000
0
6 4Bicyclo[3.1.0]hex-3-en-2-ol, 2-methyl-5-(1-methylethyl)(1.alpha.,2 .alpha.,5.alpha.)-
Scan 6 0 9 (6.571 min); K E O .D \d a ta .m s 9 1 0
/2 ..> " 2 0 30 40 5 0 6 0 70 8 0 90 100 110 120 130 140 150 160 170 180 190tf-r'K
119.1
105.1
1111 tiiL 11 ii!134.1
r jT T T T y T l ' r | t t t t j i
Abundance
900 0
800 0
7000
600 0
5000
4 0 0 0
3000
2000
1000
124950 liicyclo[3 I 0|hex :i-on-?-ol. 7-rncltiyl-O (1 mclhyldhyl). 11 alpha .2 alpha.,5.alpha )• 91.0
207 .0T m j t r
200 210
1 1 9 04 3 .0
6 5 .0109.0
27.077.0 134.0
5 3 .0
0 V-t-[ r i f i j-rr i 11
i /z -> 2 0 3 0 40 50 6 0 70T " M | M " f " " | ' TTI 1 TT-TTJTTT t J
9 0 100 110 120 130 140 150 160 170 180 190"rTTrT’ l " 200 210
77
Appendix 8 Mass Spectrum for Compound 5
Library Searched C:\Database\NIST08.LQuality 91ID .beta.-Pinene
Abundance Scan 6 8 5 (7 .0 06 min): K E O .D Id a la ms
900 0
800 0
700 0
6 0 0 0
500 0
4000
3000
2000
1000
69.1
53.1
| P 1 t i | i i I r | r i i r (I tl I | r 1 Jl I
4 1 .0
O n i j 111 r ) i i i 1 [ T M i j i i r r p T I t y t
10 20 30 4 0 50.oundance
900 0
800 0
7000
6 0 0 0
500 0
4 0 0 0
300 0
2000
1000
0
____ 207.0H ' [ ' i 111 j 1 1 1 1 j n r x | 11 11 1 1 1 1 1 [ i - r t
7 0 80 9 0 100 110 120 130 140 150 160 1 70 180 190 2 0 0 2 10//I55UO. beln I’nrnne
9 3 .0
79.0
2 7 .0
5 3 .0 121.0
15.0107.0
136.0
n T f T T T T j - T T r r i ■ ■ . i [ i i ■ I | I I M | I I I I | I I I I | I | I I | | | I I | I M I | I I ' T T J ~ T T T T y T ~ r T T ~ | ~ t ~ r T T ~ f ~ T T T T ~ f ~ T ~ T T l ] i n r . . . . . . . . . . . . . . . . . .
li -> 10 20 30 4 0 50 60 70 80 9 0 100 110 1 20 130 140 150 160 170 180 190 2 0 0 210
78
Appendix 9 Mass Spectrum for Compound 6
Library Searched : C:\Database\NIST08.L Quality : 70
: .alpha.-PhellandreneID
A b u n d a n c e
79
Appendix 10 Mass Spectrum for Compound?
Library Searched : C:\Database\NIST08.L Quality : 95ID : D-Limonene
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
6 8 1 9 3 .1
53.1
T jrm jT n T yT T n j l iL *| t t I [ - m l I 'm '
79.1
S c a n 8 5 5 (7 .9 7 9 m in): K E O .D W a la m s
107 .1121.1 136.1
rrjl tfraJ(IWt 1 4 8 .0 2 0 6 .9 2 81 .1'JTTTTyTTTTJTTTTyrTTTJT rT T pT T ri 11 i n '|T T T T [T T T r |T T ri|T T T rjT T T T [T T T rjT T IT jT T n
V z ~ > 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 .b u n d a n ce // I& 4 !t4 n I i n m n n n r -
80
Appendix 11 Mass Spectrum for Compound 8
Library Searched : C:\Database\NIST08.L Quality ! 98ID : Eucalyptol
Abundance Scan 864 (8 0 3 1 min): K E O .D W ata .m a
f I/
81
.
Appendix 12 Mass Spectrum for Compound 9
Library Searched : C:\Database\NIST08.L Quality : 97ID i Benzene, l-methyl-4-(1-methylethyl)-
f\
\ 4/
82
Appendix 13 Mass Spectrum for Compound 10
Library Searched : C:\Database\NIST08.L Quality : 90ID : 3-Cyclopentene-l-acetaldehyde, 2,2,3-trimethyl-
83
Appendix 14 Mass Spectrum for Compound 11
Library SearchedQualityI D
C:\Database\NIST08.L76Bicyclo[3.1.0]hexan-3-ol, 4 -methylene-1-(1-methylethyl)[IS-(l.alph a. ,3.beta.,5.alpha.)]-
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0l->
A bundance
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
S c a n 1 1 9 5 (9 .9 2 5 m in): K E O .D ld a la m s
7/1*4057. Bi<;ycto|3 I 0 | l i r ' , m :i-ul, 4 in c lh y le u p 1 ( In m lh y M I i y l l | I S ( I a lp h a i t w i n ! ia lp h a )|9 2 .0
8 1 0
1 0 9 .0
7 0 .04 3 .0 5 5 .0
1 3 4 0
29.0 120.0. - - 1 5 2 .0 7 \ \U 11 i i 11 | r m 11 r r 1J7 t 11 1 1 1 1 f 111111 m i »| t r i r j-ri r i f n i r j r t 111 r i n jti t i | m t t | t i i i | i i m j i i i i p i m | r ITT j n IT11 r I r 11111 f M i > f rrr t | t r r 11 t 11 11 1 n r j
* ” > 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 19Q 2 0 0 2 1 0 2 2 0 2 3 0 £ 4 0 2 5 0 2 6 0 2 7 0 2 8 0
84
Appendix 15 Mass Spectrum for Compound 12
Library Searched : C:\Database\NIST08.L Quality : 59ID : Bicyclo[3.1.1]hept-3-en-2-ol, 4,6,6-trimethyl-, [IS-(1.alpha.,2.beta.
,5.alpha.)]-
A b u n d an ce S c a n 1 2 0 9 (1 0 .0 0 5 m in): K E O D W a ta m s
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
9 4 .0
1 0 9 .0
5 9 .0
4 1 .0
7 9 .0
1 37 .0
1 5 .0 1 52 .0Or -r i i j i i n ; i i i ■ | i i i t | i | i i i > | 1 1 t r j » i >» | i i i i | i i i i | i i i i | i i i i | i i ■ i | i i i » | t i i t | i i >» | i i i ' | i i ' i | i
2 - > 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 160 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0
i 4 /
Appendix 16 Mass Spectrum for Compound 13
Library SearchedQualityID
C:\Database\NIST08.L94Bicyclo[3.2.0)heptan-3-ol, 2-methylene-6,6-dimethyl-
10001 5 2 .0
0 1 I " ' ! ■ M i ] i I I I I H H | T T I i | 111 | | 111 I I n n [ | M | ] T ' l i | i i n p m | i r 11 ,111 | M i l l m 11| | ’ , | i i M | I I I ] I 1I H I 1T i | i m ; i | | | | 1H | | I I I | | | | | | | | | | | | ? i i m i n | n n | M T i ; i > M ] | | T T ^
/Z " > 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0
86
Appendix 17 Mass Spectrum for Compound 14
Library Searched : C:\Database\NIST08.L Quality : 98ID : .alpha.-Cubebene
Abundance Scan 181 9 (1 3 .4 9 5 min); K E O .D W ata .m s
t
87
Appendix 18 Mass Spectrum for Compound 15
Library Searched : C:\Database\NIST08.L Quality : 89I D : 8 - I s o p r o p e n y l - l , 5 - d i m e t h y l - c y c l o d e c a - l , 5 - d i e n e
88
jibrary Searcnea : L:\uacaDase\iNioiuo.u Quality : 95[D : 3H-3a,7-Methanoazulene, 2,4.5,6(7,8-hexahydro-l,4,9,9-tetramethyl-, [
3aR-(3a.alpha.,4.beta.,7.alpha.))-
Appendix 19 Mass Spectrum for Compound 16
\b u n d a n c e S c a n 188 1 (1 3 .8 5 0 m in ): K E O D id a ta .m s
t ' •\ ’ '
89
Appendix 20 Mass Spectrum for Compound 17
Library Searched : C:\Database\NlSTOB.L Quality : 64ID : Naphthalene, l,2,3,4-tetrahydro-l,l,6-trimethyl-
A b u n d a n c e S c a n 1 8 8 8 ( 1 3 .8 9 0 m in ): K E O C A d a ta .m s
1 5 9 .0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000115.0 1 3 1 .0
1 7 4 0
1 5 03 9 .0
9 1 .06 5 .0
r~r i |-rri i ; i i i i | i i i i | i i r-T | rrrjr i rrp i i i | i i i rj-i i i rp i i » 11 i i i | i i t i | i i i i | i i i i | n i ' | i » ■■]■»■■ | r2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0m /z ->
Appendix 21 Mass Spectrum for Compound 18
Library Searched : C:\Database\NIST08.LQ u a l i t y
I D
A b u n d a n c e
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0m /z~ >'h u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
96Caryophyllene
S c a n 1 9 2 2 ( 1 4 .0 8 4 m in ): K E O .D V d a ta .m s
9 3 .00 0 2 2 0 1 C a ty o p tty lo n e
1 3 3 .0
4 1 .0 7 9 .0
1 0 7 .0
5 5 .0
1 4 7 .01 6 1 .0
1 8 9 .0
2 7 .0 1 7 5 .02 0 4 .0
firwrrT
on o n Af\ nr\ f in 7 n f in o n 1 0 0 1 1 0 1 9 0 1 3 0TiM| Miif» m ; n »n >TTr|m m rn |i i n n i n j >TTHi i m n Tn n i i | i n iiJ ii | i Mi | i i i n i i r r i i i i i | r T r r jm
, 0 1 4 0 1 S 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0
Appendix 22 Mass Spectrum for Compound 19
L i b r a r y S e a r c h e d
Q u a l i t y
I D
A b u n d a n c e
C : \ D a t a b a s e \ N I S T 0 8 . L
9 4
B i c y c l o [ 5 . 2 . 0 ] n o n a n e , 2 - m e t h y l e n e - 4 , 8 , 8 - t r i m e t h y l - 4 - v i n y l -
S c a n 1 9 2 2 ( 1 4 .0 8 4 m in ): K E O .D K ia ta .m s
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
9 3 .0
1 3 3 .0
7 9 .0
4 1 .0 1 0 7 .0
1 6 1 .0
5 5 .01200
1 4 8 .0
2 7 .01 7 5 .0
2 0 4 .0
O rT jm t | n 11111 n j i r n 111 n j n t ry rprrn yi riT pTTTp rir pri Tj ’ m j t ri ’ jrn * | n n j ittijit m ji i i i ; 11 M| 11 mj i m | n n p m 11n r p n j ' i j ' m |« i • ■ |i ii
m f t - > 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0
92
Appendix 23 Mass Spctrum for Compound 20
Library Searched : C:\Database\NIST08.Ljuality ID
99Azulene, 1,2,3,4,5,6,7,8-octahydro-1,4-dimethyl- 7-(1-methyletheny1)-, [IS-(1.alpha.,4.alpha.,7.alpha.)]-
M w n d a n c e
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
S c a n 1 9 5 9 ( 1 4 .2 9 6 m in ): K E O .D W a la .m s
105.0
1 4 7 .0
41.0
7 9 .0
2 0 4 .0
121.01 8 9 .0
1 6 2 0
2 6 .05 7 .0
m li->I—|—TT I T f T 1 T I | I I » I [ n 1 ' | ' I T 1 I | t ' I I | » T » » | ' I I I ' |i ii i i i i i I i ri r | i i i i | i i - i i 1 T r r r p
!20 ^ T '7o^ Vo ' 100 120 140 160 180 200 220 240 260 280 300 320 340 360
93
Appendix 24 Mass Spectrum for Compound 21
L i b r a r y Searched : C:\Database\NIST08.L Quality : 95ID : lH-Cycloprop[e]azulene, la,2,3,4,4a,5,6,7b-octahydro-l,l,4,7-tetramet
hyl-, [laR-(la.alpha., 4.alpha.,4a.beta.,7b.alpha.)]-
A b u n d an ce S c a n 1 9 8 4 (1 4 .4 3 9 m in ): K E O D \d a la .m s
3000
2000
1000
6 5 .0
18.0
m/z->i i i i ■ i n | ■ i i i | i i i ' | i i r ■ | ■ i i i | i i i ■ | i t t i- | i i i i | i i * 1 | i * i r | i t i i | i i i i | i i i * | i i i i | i i i ' | i i i i | * 1 i '*' | i
20 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0
94
Appendix 25 Mass Spectrum for Compound 22
Library SearchedQualityI D
C:\Database\NIST08.L9 8
Naphthalene, l,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-l-(l-me thylethyl)-, (1.alpha.,4a.alpha.,8a.alpha.)-
A b u n d a n c e S c a n 2 0 4 5 { 1 4 .7 8 8 m in ): K E O .D W a ta .m s
m n d an ce
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
I N a p h th a le n e . I V 3 4 4 a .5 0 , 8 a -o c la h y d iu / m c lu y l 1 m c tliy le n e I ( I m id h y le lh y l) ( I a lp h a ,4 a iip h a ,8 a a lp h a ) 1 6 1 .0
4 1 .0
9 1 .01 0 5 .0
1 1 9 .0
7 7 .0
5 5 .01 3 3 .0 2 0 4 .0
1 4 7 01 8 9 .0
1 7 5 0"I JTTfTJTTT rjTTTTJTTT'
n\li-> 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0
95
Appendix 26 Mass Spectrum for Compound 23
Library SearchedQ u a l i t y
ID
C:\Database\NlSToa.L 99Azulene, 1,2,3,5,6,7,8,8a-octahydro-l,4 -dimethyl-7-(1-methylethenyl)- , [IS-(1.alpha.,7.alpha.,8a.beta.)]-
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
S c a n 2 1 1 3 ( 1 5 . 1 7 7 m in ): K E O .D W a ta .m s
1 0 8 0
9 3 .0
7 9 .0
4 1 .0 1 3 5 01 8 9 .0
5 5 .02 0 4 .0
1 6 1 .0
1 7 5 .0
0 i r i | i i i i ] i t t r | t- t 1 i | i i i i | i i i i ; i i i i | i i i i [ i f t i ; » i i i | > i i i | i i i i ; i i i i | i i i "i | i i '~t | t r r i | ■ i i i | t
Mi-> 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0
96
Appendix 27 Mass Spectrum for Compound 24
L i b r a r y
Q u a l i t y
ID
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
S e a r c h e d : C : \ D a t a b a s e \ N I S T 0 8 . L
: 9 3
: D o d e c a n o i c a c i d
S c a n 2 2 1 9 (1 5 .7 8 4 m in ): K E O .D W a ta .m s
6 0 0
4 3 .0
1 2 9 .0
8 5 .0 1 5 7 .0
111.0200.0
mli~>2 6 .0
r. | I I T I | I T - T - l r1 8 3 .0
J I I t I | I I I I | I I I 17 '-I II | I 1 ' I I ' 1 I 'I ' I H | 11 1 I | I n I | I I I I | I I I ' | M 1 > | 1 1 1 1 I l~l ' ' I ' ' ' I I I ' ' ' |TI <
20 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
t\ \
/
97
Appendix 28 Mass Spectrum for Compound 25
Library Searched : C:\Database\NIST08.L Quality : 92ID : Caryophyllene oxide
A b u n d a n c e S c a n 2 2 9 0 (1 6 190 m in ): K E O .D W a ta .m s
4 1 .0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0L' i [ nm /z~ > <(0
7 9 .0
1 0 7 0
1 3 1 .0 1 6 1 01 8 7 0
58.0n - r p - r
60
2 0 5 .0
t i | r i i i l i i i r r t"! | i i M | i t i ’■] i i n | i ) 1 1 1 1 1 1 t i ’ [ 1 1 1 1 p i n 1 1 1 ri 1 1 1 1 1 1
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
Appendix 29 Mass Spectrum for Compound 26
Library Searched : C:\Database\NIST08.LQuality ID
A b u n d an ce
5 96-Isopropenyl-4,8a-dimethyl-l,2,3,5,6,7,8,8a-octahydro-naphthalen-2-o
S c a n 2 3 4 3 (1 6 .4 9 3 m in): K E O .D \d a la .m s
8000
7000
6000
5000
4000
3000
2000
1000
0
410187.0
105.0 2200131.0
7 9 .0
57.0203.0
i j » i t r | i i i i | Ti r i i i it i j i i i i | »m/z-> ' 20 40 6QT 80 100 120 140 160 180 200 HO 240 260 280 300 320 340 360 380 400
99
Appendix 30 Mass Spectrum for Compound 27
Library SearchedQualityID
A b u n d an ce
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000 j
1000
C :\Database\NIS1UB.l 561H-1,5 -Benzodiazepine, 2,3,4,5-tetrahydro-2-methyl-
9 1 .1
6 9 .1
51
S c a n 2 3 5 5 (1 6 .5 6 2 m in): K E O .D Id a la .m s
1 5 9 .1 2 0 $ . 1
121.1
i [ i iV r j-W 'l rlj"iJTlr l''|T I i J‘| l i f I | l i'Vt / i'^Vt |T i i' i'f i i i l'| I ' I'l | i m i ) i i i i | i ' i i | ' ' i ' | ' ' " 1 ' ' ' ' [ '
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0# 3 1 4 7 2 111 1.5 r i in z o d in z iin m o . 2 .3 4 .5 In l ia h y il io 2 nu.'lliy l
1 1 9 .0
1*
1 87 .1
■ I'llil &1 2 5 3 .0 2 8 1 .0 3 5 5 .0111' " T
1 4 7 .0
* V z - >
ju n d an ce
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
9 2 .06 5 .0
3 9 0
'z~>rj-ri i i ; l r ■ i | l i i i | i i i l 1 ■ i i i | i i i i | i n i ; i T-l i [ l i ■ i | n l i | i i i 1-|-| i 1 i | i i n ) i i l i | i i i i | rT
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
4 2 9 .17-1 I " rTT
4 2 0
" I - h - ' T4 2 0
100
Appendix 31 Mass Spectrum for Compound 28
Library S e a r c h e d
QualityI D
C : \ D a t a b a s e \ N I S T 0 8 . L
9 0
1 , 2 , 3 , 4,5 , 6 - Hexahydro-1,1,5,5 - tetramethyl - 2 , 4a-methanonaphthalen-7 ( 4 a
H)-one
Abundance S c a n 237 1 (1 6 .6 5 4 m in ): K E O .D \d a ta ,m s
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
1 7 5 .0
2 1 8 .0
1 4 7 .0
4 1 .0 9 1 .01 1 9 .0
6 9 .0
200.00 ' | ’ f » I 'JT T I r |'I~T T I I T 1 1 J TTf 1 J ! T ! I | r I rT~J1 I I 1 | I ' I 7 | I ' I 1 | I ' I I | ' ' ' | ' I I I | I ' ' > J ' T * ' | 1 1 ' ' I 1 1 I ' | I ' T ' I '
ll~> 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
101
Appendix 32 Mass Spectrum for Compound 29
Library2ualityID
M >undance
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
Searched C:\Database\NIST08.L95Isoaromadendrene epoxide
10f18 1 .1
55.1
i.
S c a n 2 3 8 7 (1 6 7 4 5 m in ): K E O D \d a la m s
147.1
M.
li
177.1
2201202)1
A i • 2 2 5 3 8 2 8 0 .9 3 2 7 .0 3 5 5 .0U‘r-TTrq-1 r t-JVtt V j W I ‘|“i i V | -Y-H i |‘i‘i i ■ t1* | i » f\ | i i i i | rn » | i i i rjr i i i ; i r t vp nrp
•ll~> 20 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0
/u n d a n c e ,77470? Is o a m n v jd m x lr c n r e |to < id .
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
4 1 .0
9 3 .0
6 7 .0
1 3 5 .0
1 6 2 .0
111.0 1 8 7 .0 2 2 0 .0
'l-> 2 0 4 0 6 0, |T" I IT-I-H-p | TT I' t | 1 T 1 1 | 1 I
1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0
102
4 0 5 .0I I | If I I
4 0 0
rrpm-r
4 0 0
Appendix 33 Mass Spectrum for Compound 30
library Searched : C:\Database\NIST08.LJuality : 94I D : l H - C y c l o p r o p [ e ] a z u l e n e , d e c a h y d r o - 1 , 1 , 7 - t r i m e t h y l - 4 - m e t h y l e n e - , [ l a R -
( l a . a l p h a . , 4 a . a l p h a . , 7 . a l p h a . , 7 a . b e t a . , 7 b . a l p h a . ) ] -
A b u n d a n c e S c a n 2 4 3 0 (1 6 .9 9 1 m in ): K E O .D V J a ta .m s
.ju n d a n c e IIW'.AO I I I <lyc lo i» io j> |<;).i /u io n e d i.c .n liy d n i 1,1 / im u M l i / l 4 im Ih y li'in . | l a K | l a n o l i n 4 a . . i l | i i ' i ! a lp l i /:■ h c ln . i’ l> . :l|iU .i )| 4 1 .0 1 6 1 .0
9000
8000
7000
6000
5000
4000
3000
2000
1000
9 3 .0
69.0
121.0204.0
1410
40 60 80 100 120 140 160i t | » r r v f- i T- r r - p 'i t r j i » » t | i r i i | i i i i | i t
180 200 220 240 260 280 300T | I T " T | T » I I | 1 I I T | » T » T f T T I r
320 340 360 380 400
f\
I /
103
Appendix 34 Mass Spectrum for Compound 31
LibraryQualityID
Searched C:\Database\NIST08.L582(10)-Pinen-3-one, (.+/-.)-
A b u n d a n c e S c a n 1 2 6 2 ( 1 0 .3 0 8 m in ): K E O .D t ia t a .m s
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
5 3 .0 8 1 .0
1 0 8 .0
4 1 .0
690
2 7 .0 1 5 0 .01 3 5 .0
93.0 1220
1 5 .0
............................................ i p r i ■ | m 11111 m 11 m 11 m 11 m 11 m 11 m 111 ■ M |i 11111 m 11 ii 111 ittjttti | ii i i | i n i 11 m | m ' l | m i [ m r i | m i i | i i i i | m i i ^
lt-> 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 - 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 07
104
Appendix 35 Mass Spectrum for Compound 32
Library SearchedQualityID
A b u n d a n c e
C:\Database\NIST08.L78B i c y c l o [ 2 . 2 . 1 ] h e p t a n - 3 - o n e , 6 , 6 - d i m e t h y l - 2 - m e t h y l e n e -
S c a n 1 2 6 3 (1 0 .3 1 4 m in): K E O .D 1 d a ta .m s
1 9 0 .9 2 0 7 .0 2 8 0 .9
m /Z ~> 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 70 180 190 2 0 0 2 1 0 220 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0A bundance *7 .1 5 0 5 B ic y c lo |7 2 l| lu ;p b in 3 o n o B ( i i l« m ilh y i-? m i'th y li)w
5 3 .0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
4 1 08 1 .0
1 0 8 .0
2 7 .06 9 .0
9 3 .01 3 5 .0
1 5 0 .0
122.0
.................................................................... , 11" " | " J" " | | " iTTT Ty n '' 1111111 1111111 i i | i i i 11 " n -p i 111 m i m i n .. ............... ............... m* * “ > 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 190 2 0 0 2 1 0 220 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0
105
Appendix 36 Mass Spectrum for Compound 33
Library SearchedQualityID
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
C:\Database\NIST08.L 96Bicyclo[3.1.11heptan-3-one, 2,6,6-trimethyl-, (1.alpha., 2 .alpha., 5.al p h a .)-
S c a n 1 2 9 4 (1 0 .4 9 1 m in ): K E O .D 1 d a ta .m s
5 5 .1
0111< 1111| |T11ITTTTTTTT
83.1
m f t - > 2 0 3 0 4 0 5 0 6 0 7 0u n d a n c e
5 5 .0 6 9 .0
9 5 .1
110.1152.1
r| n i i | l i ■ |Vl‘ l t | ll*ll| | ■ ■ . 1*1 1 . ; " " [ 1 " . I 1 1 ■ 1 I " 1 1 I ■ . I*. I . 1 . 1 I 1 " t j , I I M I | | , r , | | n 'l | ITT8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 50 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 230 2 4 0 2 5 0 2 6 0 2 7 0
II'/4041 Hicydop I l|lie|>bin .t one, 26.6 limittUtyl (1 alpha 7 ilpltn s iiph.i
8 3 .0
1 2 3 .0 1 3 7 . .I | l l ' l l t | I T T l ' |
1 6 3 .9 2 0 7 .1 2 6 6 (
9 0 0 0 '
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0 '
3 0 0 0 '
2000
1000
0
4 1 .0
9 5 .0
2 7 .0 110.01 5 2 .0
„ 1 2 3 .0 1 3 7 .00 - ' | I " 1 | I I M " " I | " I I | " " | " " ............... ... . . . I I . . . . I . . J I ; I I ■■ I . . . . I T ■ ■ ■ ! . ■ ■ . I . . . . I . . . I | I W l | , n - r r I I , 1 | I TT I | ' I I
lfc“> 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270
106
Appendix 37 Mass Spectrum for Compound 34
L i b r a r y S e a r c h e d : C : \ D a t a b a s e \ N I S T 0 8 . L
Q u a l i t y : 9 4
I D : 3 - C y c l o h e x e n - l - o l , 4 - m e t h y l - l - ( 1 - m e t h y l e t h y l ) ( R ) -
A b u n d a n c e S c a n 1 3 0 1 (1 0 .5 3 1 m in ): K E O .D ld a ta .m s
107
Appendix 38 Mass Spectrum for Compound 35
Library Searched : C:\Database\NIST08.LQualityID
64Thymol
A bundance
108
Library Searched : C:\Database\NIST08.L Quality : 86ID : 3-Cyclohexene-l-raethanol, .alpha., .alpha.,4-trimethyl-, (s)-
A b u n d a n c e S c a n 1 3 3 7 ( 1 0 .7 3 7 m in ): K E O D id a ta m s
Appendix 39 Mass Spectrum for Compound 36
>\i i
109
Library Searched : C:\Database\NIST08.L Quality : 81ID : (-)-Myrtenol
Appendix 40 Mass Spectrum for Compound 37
f
110
Appendix 41 Mass Spectrum for Compound 38
Library Searched : C:\Database\NIST08.LQuality : 98
ID : 2-Cyclohexen-l-ol, 2-raethyl-5-(1-methylethenyl)c:s-
111
Appendix 42 Mass Spectrum for Compound 39
Library SearchedDualityID
\bundance
C:\Database\NIST08.L701,8-Nonadiene, 2,7-dimethyl-5-(1-raethylethenyl)-
S u n d a n c e
9000
6000
7000
6000
5000
4000
3000
2000
1000
0
107.0#63231 I.S-NowkIichio. 2.7 (llnu:lhyl-5-(1-irolhylclliunyl)
93055.0
41.01210
69.0
149.0
135.0177.0
163.0 1920
270j r m |m r ] - f ir r p iT T |r T T i[T T r r p in |’i m | i i i i | i i n ,] iM ' j n pTnrpTTTpTTTJTTTT|TTnTTTTTTTTTT|TT?TWTTtJTTTTJTTTTTTTTT|TTTTJTffTTTTTTTTTTrpTTnTTTTyTnTJTTTf]
20 30 40 50 60 70 80 90 100110120130140150 1601701 «0190 200 210 220 230 240 250 260 270 2«f290 300 310 320 330 340
112
Appendix 43 Mass Spectrum for Compound 40
Library Searched : C:\Database\NIST08.LQuality : 93ID : Naphthalene, 1,2,3,4-tetrahydro-l,6-dimethyl-4-(1-methylethyl)(1S-
cis) -
M w n d a n c e S c a n 1 7 8 6 ( 1 3 .3 0 6 m in ): K E O ,D \d a la .m s
jndance N:i| iIiIIm Ici u 1 2,3.4-letiuliyiliol (i dmc'liyl 4-(l-inelhylnlhyl)-, ( IS asi1 5 9 .0
n /z ~ >
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
02 8 0
4 3 .0 6 3 .0 7 7 .0 9 1 .0 1 0 5 .01 2 9 .0
202.01 4 4 .0
1 8 7 .0I pTTTpTTTpTTryTrTTJTTTTJTTTTpTrrTTTTrpTrTJTTrTJITrTpTTTTTrTTpTTTnTTTTTTTTnTTn TTH")11 ii 11 nn | m 111 ir ii jTmynnjTTTr]TrTr|TTtr
2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0 3 4 0
113
Appendix 44 Mass Spectrum for Compound 41
Library Searched : C:\Database\NIST08.L Quality : 90ID : Bicyclo[7.2.0]undec-4-ene, 4,11, ll-trimethyl-8-methylene-
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
91.1
5 5 1
73)
S c a n 2 4 4 7 (1 7 .0 8 8 m in ): K E O .D V J a la m s
175.1
9 1
133 .1157.1
2 03 .1
u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0 n n i,'7~> 20
221.1
„ 2 5 2 .1 2 8 1 .0 3 2 7 .0 4 1 7 0[ » > v i"| i n r * i r i i [ 'l m i | H n ’ T it r p r n i | » 11 ■ j f n | -p ’ ' t‘f*| i i i »1 i r r r p - ' i i i | i i i i j i n t j ■ 1 i » | i i r r | > i > i | i i i i | I T
2 0 4 0 6 0 8 0 1 0 0 1 2 0 140 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0
9 3 .0#62375 Bic:yclo[7.2 (l)unduc4 4,11.11 liiiiii>tliyl-8 m«tliyleiip
6 9 0
4 1 01 3 3 .0
1 6 1 .0
1 8 9 .0
1 1 5 .011 l l ' l ' l ' l 11 I 11 I 11 11 1 I I 1 | ■ l i i | ■ r r T | M i i | i i i i | i i i i | i M i | M i i | ^ M i | i i i i | i i i ' [ i ( i i | i — 11 | i i i i~| i i i i | i
40 60 80 100 120 140 160 180 200 220 240 260- 280 300 320 340 360 380 400 420
Appendix 45 Mass Spectrum for Compound 42
LibraryQualityID
Searched C:\Database\NIST08.L533,7-Cyclodecadien-l-one, 3,7-dimethyl-10-(1-methylethylidene)(E,E)
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
9 5 .1
S c a n 2 5 0 5 ( 1 7 .4 2 0 m in ): K E O .D \d a ta .m s 220.2
5 5 .1
77
1 2 3 .1
-» i i f i t i i | > 'Lii | r r t t - j t t' t i j I > t i j ? i t t y r r i » p t'n | V v r V - j - r f 1 i‘ H 1 i t ‘ f | i i i i | i i n p m | t > r l y r i i r j > t i r p t t i p » » i - | - n i i p
2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 34C 3 6 0 3 8 0 4 0 0 4 2 0/1 7 3 2 5 0 : ( , / C y c k x Ic c .ii lH n I o w 3 , / d im e th y l 10 (1 m o th y lu M iy b li in e )- . ( I I I- 1 0 7 .0
1 3 5 .0
1 59 .1
191 .1
',.1 J ,iW-ri- A M .2 2 8 1 0 3 3 1 .0 3 5 5 .0 4 0 5 .1 4 2 9 .1
iz->ju n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
O’
6 7 .0
4 1 .0
V l~ >
1 8 .0 8 9 .0T f l | ? I I I ] T I I l*[TT»
1 7 5 .0 2 1 8 .0
1 5 7 .0 2 0 0 .0' " " " I " " I " ' ’ I " " I ' " ' I ' " ' I" "I " " I " I "FT! | " " I " " P 111 r
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
115
Appendix 46 Mass Spectrum for Compound 43
Library SearchedQualityID
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
C :\Database\NiSTU8.l 86l-Pyrrolin-2-amine, N-(1-adaraantyl)-
S c a n 2 5 1 8 (1 7 .4 9 5 m in ): K E O .D \d a ta .m s 216.2
105 .1 1 47 .1
55.179.1
•'Z"> 20.u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0Vz-> 20
I [ ■ 11"i *| t r i- | i V 'i i 'i ' i I i< ] i i 1 i ! 1 i I V 1 I H - I " I | i n i f -m i | i m | i i > ' | " " I ' " ' T -’ " r 1^
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0,7 /.1 1 ilb 11’yifGlin 7 n m im ' N ( la d a m n n ly l )
8 5 .0 2 1 8 .0
1 >3.1
i"!
175 .1
2 3 6 2 2 5 4 .0 2 8 1 .0 3 3 1 .1 3 5 5 .0
4 1 .0
1 6 1 .0
1 3 5 .0
6 7 .0
1 0 7 .01 9 0 .0
T i t u i > | u n j r m [ » I ' r r y T T i T f ' i t n j 1 1 ? i 11 r i t j - i i j > | i 1 1 i j t i r i | j r i i i i t t i f i f i i | > i t r 1 1 1 i i j i i ' i | i t
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 6 2 8 0 3 0 0 3 2 0 3 4 0 ■ 3 6 0 3 8 0
4 0 4 9 4 2 8 .9M " " I' ' ' 114 0 0 4 2 0
f I | f I I 1 | I » IT |
4 0 0 4 2 0
116
Appendix 47 Mass Spectrum for Compound 44
Library Searched : C:\Database\NIST08.L Juality : 86ID : 2H-Cyclopropa[a]naphthalen-2-one, l,lai4,5,6,7,7a,7b-octahydro-l,l,7,
7a-tetramethyl-, (la.alpha.,7.alpha.,7a.alpha.,7b.alpha.)-
M ju n d a n c e S c a n 2 5 3 8 (1 7 .6 0 9 m in ): K E O .D \d a ta .m s
'lUundance 473272 21 lCyr.lo|>to|w|;i)miphlli;ili n 2 one 1.1.1.4.M i.7 ,/i 7b-o«d.«hyilro-1 : V 7:i tulmiiwthyl. (In .ilpln ' tu . ilc n ' I i .iM . i
2 1 8 .0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
41.0
7 7 .0
1 4 7 .0
1 1 9 .01 7 5 .0
9 6 .0
U ‘T j n
*"> 207 I T T I I I I
4 0 6 0, m i r t T - i - r r r T - r T | ,
1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 ? 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
117
Appendix 48 Mass Spectrum for Compound 45
Library SearchedQualityID
A b u n d a n c e
C:\Database\NIST08.L94Caryophyllene-(II)
S c a n 2 6 2 6 ( 1 8 .1 1 3 m in ): K E 0 .D 1 d a ta .m s
- ' z ~ > 2 0 40jn d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0#62301 Caryophyllene il l)
1 0 5 .0
1 3 3 .0
4 1 .0
1 6 1 .0
7 9 0 1 8 9 .0
r r m r > t t t t r m r r r r r r | T » I I | r I T T | I T T'T [ T
<Z-> 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
/
118
Appendix 49 Mass Spectrum for Compound 46
LibraryQualityI D
\b u n d a n c e
Searched C:\Database\NIST08.L91Alloa romadendrene oxide-(1)
S ca n 2 6 9 6 (1 8 .5 1 3 m in ) K E O .D W a ta .m s
jn d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
V47/5 Alluaromfidewlfoni! oxide (l)4 1 .0
9 1 .0
6 9 .0 1 0 9 .0
1 3 5 0 , 7 7 -0
1 5 9 .0202.0220.0
r i | 7 » I I | I T TT I ' I I I I | I I I 1 [ I 1 r T | I I I I | 11 I I | I >1 I | I M l [-1 I I H I I I I | I I I 1 | I TT I | t I 1 I | I I I I | TTI I | I T
m/z~> 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
119
Appendix 50 Mass Spectrum for Compound 47
Library Searched : C:\Database\NlSTOB.b Quality : 90ID : Culmorin
A b u n d a n c e S c a n 2 7 7 3 ( 1 8 .9 5 4 m in ): K E O .D V Ia ta .m s
L
1 2 0
Library Searched : C:\uacaDase\wiSTuo.L Quality : 47ID : cis-Z-.alpha.-Bisabolene epoxide
Appendix 51 Mass Spectrum for Compound 48
A b u n d a n c e S c a n 2 7 6 6 (1 9 .0 2 8 m in): K E O D V Ja ta .m j
t\
\|/
121
Appendix 52 Mass Spectrum for Compound 49
Library Searched : C:\Database\NIST08.L Quality : 83ID : 5-l8opropenyl-2-methyl-7-oxabicyclo(4.1.0]heptan-2-ol
A b u n d a n c e S c a n 2 8 2 2 ( 1 9 .2 3 4 m in ): K E O .D \d a ta .m s
u n d a n c e4 3 .0 9 5 .0
(U57I7: !> ls(i|»0|)L'iiyl 2 nv.lh/l / iwabit/i lo|4 I (ijlmpt.in 2 ul
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0 7 1 .0
3 0 0 0
2000
1000 12202 6 0 1 5 0 0 « * „ n
1 6 8 0( r ~ r j T r T T J T T T r j n I i ; i m I I i r r | i » i f ] l » i t j t v i 1 [ I r r I I t I i > ] I i « i p r r T J T T T T J T T T T J T T T T T T T T T T T T r r r p j - r - i [ i
'z ~ > 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
122
Library Searched : C:\Database\NIST08.L Quality : 89ID : Tricyclo[4.3.0.0(7,9)]nonane, 2,2,5,5,8,8-hexamethyl-( (1.alpha.,6.be
t a .,7.alpha.,9.alpha.)-
Appendix 53 Mass Spectrum for Compound 50
f\ t
123
Library Searched : C:\Database\NIST08.L Quality : 78ID : Longifolenaldehyde
Appendix 54 Mass Spectrum for Compound 51
A b u n d a n c e S c a n 2 9 0 0 ( 1 9 .6 8 0 m in ): K E O .D Id a ta .m s
124
Appendix 55 Mass Spectrum for Compound 52
LibraryQualityI D
Searched C :\Database\NIST08.L 83lH-Inden-l-ol, 2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-
A b u n d a n c e S c a n 3 0 8 9 (2 0 .7 6 2 m in ): K E O .D W a la .m s
1 7 5 1
'z->u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
-p -» i i‘ | t » TT*fT 'H t y T ’n ' r p 'T iT 'f f >1 f 7*1*1 p T f f j - r r r r p fT r p f n p r i i i | r r r i |— t r f | i i i i-|t i > i | i2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0
//4 4 ! )5 1 . I l l liv li.'ii I i:l yt.'iH.'i / a l ic M l iy d iu 4 . ' l 7 a t io ic lh y l 1 4 7 .0
1 6 5 .0
4 3 .0 1 2 3 .0
9 5 .0
6 9 .0
2 7 .0
1 8 1 .0
T T f T T T T J T T n q I I f I J I T T 7 • T! 1 I [ ' T 1 T | I I T T J T T 7 T | T IT T [ T i l r p T T t T [ T T T T y T I T ! | T T T~rJ r I
2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0
4 0 0 .8I I | ITT
4 0 0
T - r p - r r
4 0 0
Library Searched : C:\UataDase\iusTuo.ij
Appendix 56Mass Spectrum for Compound 53
Quality ID
9 21H-Cycloprop[e]azulene, la,2,3,5,6,7,7a,7b-octahydro-l,1,4,7-tetramet hyl-, [laR-(l a .alpha.,7.alpha.,7a.beta.,7b.alpha.)]-
A b u n d a n c e S c a n 3 6 1 7 (2 3 .7 8 3 m in ): K E O .D ld a ta .m s
a b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
0
«62Mf» I I I Cyi.k)|iiO()(i:):i/lileiw l.i 2.3 f»l>./.7a 71) ix.laliy.lnil I <| / It'll niuHtiyl |lnH (1,i i . / .ilpli.i ./■> 71. ilyiii i i|4 1 .0 1 0 7 .0
1 6 1 0
7 9 .01 3 5 .0
2 0 4 .0
6 0 .0 1 6 2 .0i TTTTi tTT-]-r» 11| i m [ h i 1111 111 111111 111111 r 11 n f r| 11 1111 h i | m it | f i fr|1 f r r p ■ » m r | 11 i tj 1 1 »i | i i i i | » r
• /z ~ > 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0
126
Library Searched : C:\Database\NIST08.L Quality : 50ID : Methyl 4,6-decadienyl ether
Appendix 57- Mass Spectrum for Compound 54
A b u n d a n c e S c a n 3 8 8 8 (2 5 .3 3 4 m in ): K E O .D W a la .m s
9 0 0 0
6 0 0 0
7 0 0 0
6 0 0 0
6 0 0 0
4 0 0 0
3 0 0 0
2000
1000
6 9 .0
9 7 .0
1 4 0 .01 6 8 .0
' z - > 4 0 6 0 8 0
1 1 7 .0
1 2 0 1 4 0 160
I I f T T
1 8 0 2 0 0
II H [ • T
220 2 4 0ITT | " 111 rrr iM| ,
2 6 0 2 8 0 3 0 0 3*7i i m i r p n »|i i i f | n i n i it t i i n r f n »11 ' i n 11 n i
3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0
127
Appendix 58Mass Spectrum for Compound 55
Library Searched : C:\Database\NIST08.L Quality : 99ID Dodecanoic acid, tetradecyl ester
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
S c a n 4 1 4 5 (2 6 .8 0 4 m in ): K E O .D 1 d a ta .m s 20)2
57.1
8 3 1
111.1
.. W |J lil11r[ i t*11 p i i t | n i------------
168.12 2 9 .2
3 9 6 .4l A l l l i . J l i i . L ....... I . L . .. L l 2 5 6 2 2 81 0 3 1 1 .1 3 4 1 .0 3 6 8 .2 [ ' 4 2 9 .1 4 5 0 .9 4 7 9 .0 5 0 4 .011 rr i-r11 iVrp-rrrp i'rV-fT i i r ;«■ r¥i i p m 11 m fftr i f-i i n 111 n 1111 f| n 11 pm ■ | n 1111111 j m 11111 r
'Z-> 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3C 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0jn d a n c e # 1 9 4 3 3 3 D o d c c . i t w . , k:kI lo lM fk ii.y l k s I o i
201.0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0 5 7 .0
5 0 0 0
4 0 0 0 8 3 .0
3 0 0 0
2 0 0 0 2 8 0 111 0
1000 1 6 8 .0
z -->
1 4 0 .0 2 4 1 0 3 9 6 0' 2 6 9 .0 2 9 7 .0 3 2 5 .0 3 5 3 .0
0 T I IT f I r | r M 11 r IT 1 | I I m j T * • • • I T 1 T J T i i ] t t t 1-j I r r r p t n | m r p m t |ttt tjitt r f ^ r r f " - tt j i r r q i ttt jtt i i jtittjttttj i - r r r j n r t j t t t r j t- t-t t t
2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0
7\
\|/
128
Appendix 59 Mass Spectrum for Compound 56
Library Searched : C:\Database\NIST08.L Quality : 99ID Dodecanoic acid, hexadecyl ester
A b u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
S c a n 4 3 9 1 (2 8 .2 1 2 m in ): K E O .D W a ta .m s201.2
57.1
oUprrrjTT
9 7 .1
'z ~ > 2 0 4 0u n d a n c e
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
125 .11 57 .1
2 2 4 .2
2 6 9 .24 2 4 4
I , , , . . W \ I. , 2 9 7 .2 3 1 9 2 3 4 1 .0 3 8 1 .3 j 451 0 4 7 9 .1 5 0 3 .1 5 2 9 .2TTrrf m i | ' t i*r j rrr- |4 tt t j r*-rr|-n~rr|*T m t 11 n i p ' it | r t n ■ h t t ji i rr-j- i m j i r n p n t p-rrr yn ....................................................................8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0
//2 D 2 2 5 8 D n d u c a n o ic a c id , h e x m lc c /l c i lo i 201.0
5 7 0
8 3 .0
111.02 2 4 .0
1000 2 8 .0
1 40 .01 6 8 .0 2 6 9 .0
4 2 4 .0
2 4 6 .0 2 9 7 0 3 2 5 .0 3 5 3 .0 3 9 6 .0t r r | 1 ' ' IJ iT r * 11T11J t r r r p - r r r j r i v r | r »n y r m y t i m j l x r r p r r r p T i n t f r r p rTTJTTTTJTTTTTTTTT| i m p r r
'z - -> 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 ^ w I o O ^ V m
129
Appendix 60 Mass Spectrum for Compound 57
Library Searched : C:\Database\NIST08.L Quality : 72ID : Succinic acid, heptyl tridec-2-ynyl ester
A b u n d a n c e S c a n 4 5 1 4 (2 8 9 1 6 m in ): K E O .O \d a ta .m s
u n d a n c e i/ l '. t .< 4 /8 S u c c in ic ai'id, in plyl Iih Ihc ? v n y leslur
101.0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
2000
1000
O'
5 7 .0
2 9 .07 9 0
1 2 3 .0 1 4 9 .01 9 9 .0
rTTTfTTTTJ"2 4 7 .0 2 7 8 .0
TTJ T111 J I T T T-y T -p-r I t 11 t i t 111111111 r | M 11 p r
ii~> 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 '3 8 0 4 0 0 4 2 0 4 4 (7 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0
130
Appendix 61 Mass Spectrum for Compound 58
Library Searched : C:\Database\NIST08.L 3uality : 98ID : Dodecanoic acid, octadecyl ester
t\j t
131
Appendix 62 Mass Spectrum for Compound 59
Library SearchedQualityI D
C : \ D a t a b a s e \ N I S T 0 8 . L
7 0
L o n g i p i n o c a r v e o l , t r a n s -
A b u n d a n c e S c a n 4 4 6 2 (2 8 .6 1 6 m in ): K E O .D \d a ta .m $
4 1 .0
9 0 0 0
8 0 0 0
7 0 0 0
6 0 0 0
5 0 0 0
4 0 0 0
3 0 0 0
6 9 .0
9 5 0
1 1 8 .01 5 9 .0
2000
1000
1 8 7 .0 220.0
' z - >
0 t i | rrnj rr2 0 4 0
rjTT-TTjTTi I | r n r ; i m p i r r p i i i| i i i i f . 1J ’ | ■»■' P » 11| r i n 111 n |m i p i i r n i n ytm | n rt |T'i i > [m n i » i n r i » ttj1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0
f\
\ /
132