ethnobotanical uses, phytochemical analysis, bioactivity

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 10 Mass Spectrum for Compound7 80













Appendix 23 Mass Spctrum for Compound 20 93




Appendix 27 Mass Spectrum for Compound 24 f • 97\ ,

Appendix 29 Mass Spectrum for Compound 26 I 99


Appendix 1 Questionnaire used for traditional medicine practitioners 68

ix

























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




x


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.

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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

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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.

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Asma J., Ghulam M. S., Shabnum S., Amin S., Abdul N., .. - nt ■ •

Sarfaraz K. M. (201 l).Chemotaxonomic clarification of pharmaceutically important

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amino acids analysis of Cyperus articulatusL.(Cyperaceae) extracts and the effects of the

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!° E B., Schmutz M., Meyer C., Bopelet M., Portet C., Jeker A.,.Rakotonirina S.V, H.R. Olpe,

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(Cyperaceae).Journal of Ethnopharmacology. 76.145-150

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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.

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ithC. D., Shannon F., Evangelos B., Melony M. and Valerie B.

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H 1 S. R., Elisabeth N. B., Alice R., Merc B. (2000). Sedative properties of the decotion of the

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66

jHO (2003). WHO guidelines on good agricultural and collection practices (GACP)

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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


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


• ' *Vl ' '\ »

76


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


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


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


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

.


Library Searched : C:\Database\NIST08.L Quality : 97ID i Benzene, l-methyl-4-(1-methylethyl)-

f\

\ 4/

82


Library Searched : C:\Database\NIST08.L Quality : 90ID : 3-Cyclopentene-l-acetaldehyde, 2,2,3-trimethyl-

83


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


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 /


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


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


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.))-


\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


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 ->


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


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


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



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


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


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


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


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



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


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


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


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


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



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



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


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


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


>\i i

109

Library Searched : C:\Database\NIST08.L Quality : 81ID : (-)-Myrtenol


f

110


Library Searched : C:\Database\NIST08.LQuality : 98

ID : 2-Cyclohexen-l-ol, 2-raethyl-5-(1-methylethenyl)c:s-

111


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


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


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


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



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


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



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


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


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


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.)-


f\ t

123

Library Searched : C:\Database\NIST08.L Quality : 78ID : Longifolenaldehyde


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


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


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


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


Library Searched : C:\Database\NIST08.L 3uality : 98ID : Dodecanoic acid, octadecyl ester

t\j t

131



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

ethnobotanical uses, phytochemical analysis, bioactivity

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