CHEMICAL INVESTIGATION OF OPUNTIA SPECIES AND STENOCEREUS THURBERI, CACTI NATIVE TO NORTH AMERICA: A SEARCH TO IDENTIFY ESSENTIAL OIL COMPOSITIONS AND TO CHARACTERIZE CYCLIC SILOXANE COMPOUNDS
by
CYNTHIA RENA WRIGHT
A DISSERTATION
Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosopy in The Biotechnology Science and Engineering Program to The School of Graduate Studies of The University of Alabama in Huntsville
HUNTSVILLE, ALABAMA
2013
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In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree from The Uiiiversity of Alabama in Huntsville, I agree that the Library of this University shall make it freely available for inspection. I hrther agree that permission for extensive copying for scholarly purposes may be granted by my advisor or, in hislher absence, by the Chair of the Department or the Dean of the School of Graduate Studies. It is also understood that due recognition shall be given to me and to The University of Alabama in Huntsville in any scholarly use which may be made of any material in this dissertation.
(student signature) (date) DISSERTATION APPROVAL FORM
Submitted by Cynthia Rena Wright in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Biotechnology Science and Engineering Program and accepted on behalf of the Faculty of the School of Graduate Studies by the dissertation committee. We, the undersigned members of the Graduate Faculty of The University of Alabama in Huntsville, certify that we have advised and/or supervised the candidate on the work described in this dissertation. We further certify that we have reviewed the dissertation manuscript and approve it in partial hlfillment of the requirements for the degree of Doctor of Philosophy in the Biotechnology Science and Engineering Program.
Committee Chair n (Date)
Cha
j 1 / (1I / fl College Dean \ I 5 \ 3 Graduate Dean
iii ABSTRACT '
The School of Graduate Studies The University of Alabama in Huntsville
Degree Doctor of Philoso hy Program Biotechnology Science and Engineering-
Name of Candidate Cynthia Rena Wright Title Chemical Investigation of Opuntia species and Stenocereus thurberi, Cactus Native to North America: A Search to Identify Essential Oil Compositions and Characterize Cyclic Siloxane Compounds
This study investigates the essential oil compositions of certain cacti species native to southern Arizona and the California Channel Islands. The species studied are Opuntia littoralis (Engelm.) Cockerell., Opuntiaficus-indica L. (Mill), Opunita prolifera Engelm., Opuntia acanthocarpa var. major (Engelni. & J.M. Begelow) L.D. Beiison, Opuntia phaeacantha var. discata, and Stenocereus thurberi Engelm. Drosophila mojavensis, a cactus fly, utilizes the necrotic rots of 0. littoralis and S. thurberi, which have been shown to be toxic to fruit flies, Drosophila melanogaster. The oil compositions of both 0. littoralis and S. thurberi was characterized at differing states of necrosis and compared to laboratory rot specimens. Toxicity analysis was performed 011 Drosophila melanogaster to ascertain toxicity to individual volatile compounds identified in the necrotic cactus rots. Specimens of 0. littoralis, 0. ficus-indica, and S. thurberi were examined for the presence of cyclic oligosiloxane compounds. These cyclic compounds were characterized by gas chromatography-mass spectrometry. Confocal niicroscopy was employed to identify the location of silica bodies in the cactus tissues.
Abstract Approval: Committee Chair
Graduate Dean
CONTENTS
Page
List of Figures………………………………………………………………. viii
List of Tables……………………………………………………………….. xi
Chapter
1. INTRODUCTION
1.1 Purpose of the Study…………………………………………... 2
1.2 Santa Catalina Island, California Opuntia Species……………. 4
1.3 Arizona-Sonora Desert Opuntia Species……………………….. 9
1.4 Stenocerus thurberi Engelm. growing at Organ Pipe Cactus
National Monument…………………………………………….. 13
1.5 Necrotic Opuntia littoralis (Engelm.) Cockerell an Stenocereus
thurberi Engelm.……………………………….……………….. 15
1.6 Silica Bodies in Cactus……………………………………...... 17
2. MATERIALS AND METHODS
2.1 Plant Collection and Extraction………………………………… 19
2.1.1 Plant Collection and Extraction of Santa Catalina
Island Califonia Opuntia Species……………………………….. 20
2.1.2 Collection and Extraction of Arizona-Sonora
v
Desert Museum Opuntia Species……………….………………. 21
2.1.3 Collection and Extraction of Stenocereus thuberi Engelm….. 22
2.1.4 Collection and Dynamic Headspace Extraction of
Necrotic Opuntia littoralis (Engelm.) Cockerell and
Stenocereus thurberi Engelm………………………………….. 22
2.2 Microscopic Investigation…………………………………….. 25
2.3 Chromatographic Identification of Compounds…………...... 25
2.4 Carboxylic Acid Esterification………………………………... 26
2.5 Fly Toxicity Testing…………………………………………… 27
2.6 Drosophila melanogaster Olfactory Preference Test…………. 27
2.7 Structure Determination………………………………………. 28
2.8 Single-Crystal X-Ray Diffraction…………………………… 28
3. RESULTS
3.1 Volatile Compounds Identified in Santa Catalina Island,
California Opuntia Species……………………………………… 30
3.2 Volatile Compounds Identified from Arizona-Sonora Desert
Opuntia Species………………………………………………… 47
3.3 Essential Oil Composition of Green Stenocereus thurberi……. 64
3.4 Volatile Compounds Identified in Necrotic Opuntia littoralis
and Stenocereus thurberi………………………………………. 67
vi
3.5 Toxicity and Olfactory Preference of Drosophila
melanogaster to Cactus Volatiles………………………………. 92
3.6 Characterization of Silica Bodies in O. littoralis,
S. thurberi, and O. ficus-indica...... 97
4. DISCUSSION
4.1 Santa Catalina Island, California Opuntia Species……….. 118
4.2 Arizona-Sonora Desert Opuntia Species………………...... 119
4.3 Stenocereus thurberi Englem. Growing at Ogan Pipe
Cactus National Monument……………………………….... 120
4.4 Necrotic Opuntia littoralis and Stenocereus thurberi……… 120
4.5 Characterization of Cyclic Oligosiloxanes in O.
littoralis, O. ficus-indica, and S. thurberi………..………… 122
5. CONCLUSION
5.1 Findings…………………………………………………….. 126
5.2 Future Work…………………………………………...... 127
APPENDIX…………………………………………………………………. 129
WORKS CITED……………………………………………………………… 140
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LIST OF FIGURES
Figure Page
1.1 Photograph of Santa Catalina Island, California………………………. 4
1.2 Photograph of coastal cactus wren (Campylorhynchus brunneicapillus) perched on O. littoralis……………………………….. 6
1.3 Photograph of Opuntia littoralis (Engelm.) Cockerell………………….. 8
1.4 Photograph of Opuntia ficus-indica L. (Mill)…………………….….... 9
1.5 Photograph of Opuntia prolifera Engelm……………………….…….. 9
1.6 Photograph of Arizona-Sonora Desert Museum………………………. 10
1.7 Photograph of Opuntia acanthocarpa var. major (Engelm. & J.M.
Bigelow) L.D. Benson…………………………………………………. 13
1.8 Photograph of Opuntia phaeacantha var. discata (Griffiths) L.D.
Benson & Walk………………………………………………………… 13
1.9 Photograph of Stenocereus thurberi Engelm...... 14
2.1 Photograph of Lickens-Nickerson apparatus…………………………… 21
2.2 Photograph of dynamic headspace extraction apparatus..…..…………. 27
3.1 Structures of some compounds identified in the Santa Catalina Island,
California species……………………………………………………….. 45
3.2 Structures of some compounds identified in the Arizona Sonora
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Desert species…………………………………………………………… 63
3.3 Photograph of early field rot O. littoralis……………………………… 68
3.4 Photograph of exterior natural rot O. littoralis………………………… 68
3.5 Photograph of late rot O. littoralis…………………………………….. 68
3.6 Photograph of early rot S. thurberi……………………………...... 76
3.7 Photograph of late rot S. thurberi……………………………………… 76
3.8 Micrograph of O. littoralis…………………………………………….. 98
3.9 Micrograph of Crystal removed from O. littoralis……………………. 98
3.10 Micrograph of S. thuberi………………………………………………. 98
3.11 Micrograph of crystal removed from S. thurberi……………………… 98
3.12 Micrograph of stained O. littoralis .……………………………….. 102
3.13 Micrograph of stained S. thurberi………………………………….. 102
3.14 Micrograph of stained O. ficus-indica……………………………… 102
3.15 Micrograph of stained O. ficus-indica…………………………...... 102
3.16 Gas Chromatogram of green necrotic S. thurberi……..…………... 103
3.17 Gas Chromatogram spectra of S. thurberi peak 44.955.………...... 110
4.1 Gas Chromatogram of Column Bleed………………………………….. 124
6.1 Gas Chromatogram of O. littoralis with silica bodies……….………… 129
6.2 Gas Chromatogramof O. littoralis lacking silica bodies……….……… 129
6.3 Gas Chromatogram of O. ficus-indica………….……………………… 130
6.4 Gas Chromatogram of Opuntia prolifera growing on Santa Catalina
Island, California….…………………………………………………….. 130
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6.5 Gas Chromatogram of Opuntia acanthocarpo var. major growing at
Arizona-Sonora Desert Museum………………………………………… 131
6.6 Gas Chromatogram of Opuntia acanthocarpa var. major growing in
Organ Pipe Cactus National Monument………….…………………….. 131
6.7 Gas Chromatogram of of Opuntia phaeacantha var. discata growing
wild at Arizona-Sonora Desert Museum……..…………………………. 132
6.8 Gas Chromatogram of Opuntia phaeacantha var. discata growing
wild at Organ Pipe Cactus National Monument…………..……………. 132
6.9 Gas Chromatogram of lab rot O. littoralis…………..………………… 133
6.10 Gas Chromatogram of Stenocereus thurberi necrotic dynamic
headspace extraction…………………….……………………………… 133
6.11 Gas Chromatogram of necrotic Stenocereus thurberi early rot………. 134
6.12 Gas Chromatogram of late, natural rot S. thurberi…………..……….. 134
6.13 Gas Chromatogram of green rot S. thurberi………………...... 135
6.14 Gas Chromatogram of lab rot S. thurberi……………..……………… 135
6.15 1H NMR spectra of Opuntia littoralis crystalline powder………...... 136
6.16 1H NMR spectra of Stenocereus thurberi crystalline powder.……….. 136
6.17 13C NMR spectra of Opuntia littoralis crystalline powder…………… 137
6.18 13C NMR spectra of S. thurberi crystalline powder…………………. 137
6.19 D. melanogaster toxicity raw data…………………………………….. 138
x
LIST OF TABLES
Table Page
3.1 Essential oil components of Opuntia littoralis………………………… 32
3.2 Essential oil components of Opuntia ficus-indica……………………… 38
3.3 Essential oil components of Opuntia prolifera………………………… 42
3.4 Essential oil yields of Arizona Opuntia species………………………... 47
3.5 Chemical composition of the essential oil of Opuntia
acanthocarpa var. major from Organ Pipe Cactus National
Monument…………………………………………………………. 51
3.6 Chemical composition of the essential oil of Opuntia
acanthocarpa var. major from Arizona-Sonora Desert
Museum…………………………………………………………….. 54
3.7 Chemical composition of the essential oil of Opuntia
phaeacantha var. discata from Organ Pipe Cactus National
Monument…………………………………………………………… 57
3.8 Chemical composition of the essential oil of Opuntia
Phaeacantha var. discata from Arizona-Sonora Desert
Museum……………………………………………………………… 60
3.9 Chemical composition of green Stenocereus thurberi Engelm. obtained
from Organ Pipe Cactus National Monument………………………... 64
3.10 Essential oil composition of lab-rot Opuntia littoralis………………….. 70
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3.11 Volatile composition of early natural rot S. thurberi Engelm………….. 77
3.12 Essential oil composition of field-rot Stenocereus thurberi Engelm…… 81
3.13 Essential oil composition of lab-rot S. thurberi………………………… 86
3.14 Toxicity of D. melanogaster to certain compounds identified in
Cactus rots……………………………………………………………. 92
3.15 Oil recovered from hydrodistillation of green Opuntia cactus species
And presence of cyclic oligosiloxanes……………..………………… 104
3.16 Oil recovered from necrotic O. littoralis, green S. thurberi, and
Necrotic S. thurberi and the presence of cyclic oligosiloxanes……… 106
3.17 Volatile cyclic siloxane composition of O. littoralis and S. thurberi…. 108
3.17 Volatile composition of Opuntia littoralis lacking silica bodies……… 112
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CHAPTER 1
INTRODUCTION
The work for this dissertation is three-fold. The first part of this dissertation is the characterization of volatile compounds from five species of Opuntia cactus and one species of Stenocereus cactus. Hydrodistillation and gas chromatography-mass spectrometry (GC-MS) were used for the characterization of volatile compounds. Some cactus species such as S. thurberi may live for hundreds of years in the extreme heat and arid conditions of the desert. How plants adapt to this severe climate may lead to a better understanding of how other plant species may respond to climatic changes in the future.
The necrotic rots of two of these cactus species are the food source for a species of cactophilic fly, Drosophila mojavensis. The second part of this dissertation was the characterization of the volatile fraction of the necrotic rots of Opuntia littoralis and
Stenocereus thurberi using dynamic headspace extraction, hydrodistillation, and GC-MS.
During the characterization of the essential oil of Opuntia littoralis, Opuntia ficus-indica, and Stenocereus thurberi the presence of cyclic oligosiloxane within the biomass of each of these three species of cactus was suggested. The third part of this dissertation is the
characterization of oligosiloxane compounds by gas chromatography-mass spectrometry.
Additionally, specimens of cactus were subjected to compound and confocal microscopy.
1.1 Purpose of this Study
Cacti are native to the Americas and can be found growing in diverse regions from Patagonia to Western Canada. Cacti typically inhabit environments that suffer from extreme drought. Cacti lack photosynthetic leaves. Instead, they have spiny needles and photosynthetic stems. Cacti are classified within the plant family Cactaceae and subdivided into two subfamilies: opuntias (subfamily Opuntioideae) and cactoids
(subfamily Cactoideae). The cactus species investigated in this study include members of the Opuntia subfamily growing on Santa Catalina Island, California-Opuntia littoralis
(Engelm.) Cockerell., Opuntia ficus-indica L. (Mill), and Opuntia prolifera Engelm-and the following Opuntia species growing in the Sonora Desert of Arizona: Opuntia acanthocarpa var. major, and Opuntia phaeacantha var. discata. A single member of the cactoid family-Stenocereus thurberi Engelm., which is native to the Sonora Desert of southern Arizona, was also studied. None of these cacti has previously been subjected to essential oil analysis. The volatile composition of these species is unknown.
Necrotic rots of O. littoralis (Engelm.) Cockerell and S. thurberi Engelm. are utilized as a food source by a cactus fly, Drosophila mojavensis. These rots are lethal to the common fruit fly, Drosophila melanogaster. D. mojavensis evolved into four geographically isolated subspecies that colonize the Mojave Desert, the Sonoran Desert and Baja California. The essential oil compositions of the necrotic rots of O. littoralis
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(Engelm.) Cockerell and S. thurberi Engelm. are investigated in this study to determine which major components identified in the essential oils are toxic to the fruit fly. No previous study has characterized the essential oil composition of these necrotic rots. The cactus flies rely on the biomass of the cactus rots, which contains essential oil, for sustenance. Identifying the composition of the essential oil of these cactus rots is a fundamental step in studying the evolution of Drosophila from a consumer of fruit to a consumer of necrotic cactus.
Silica bodies were previously identified in S. thurberi Engelm and other columnar cactus. The role of silica bodies in plants has yet to be determined. Although industrially produced silica bodies have been studied, silica bodies within cactus have only been subjected to hydrofluoric testing and observation by compound microscopy. The purpose of this study was to determine whether silica bodies were also present in O. littoralis and
O. ficus-indica. GC-MS analysis of extracts of the essential oils of O. littoralis, O. ficus- indica, and S. thurberi suggested that these cactus contained cyclic siloxanes. This study sought to verify the presence of large, cyclic siloxanes within the cacti species studied.
This chapter introduces the different species of cactus studied, the importance of certain cactus rots to the survival and evolution of a species of cactus fly, and gives an overview of the previous identification and characterization of silica bodies in certain cactus species.
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1.2 Santa Catalina Island, California Opuntia Species
Santa Catlina Island is one of a handful of barrier islands located off the coast of
Southern California (Figure 1.1). Santa Catalina Island is a one-hour boat ride from Long
Beach California, which is located in the Los Angeles Metropolitan area.
Figure 1.1 Google map image of the California Channel Islands. Note Santa Catalina Island, California is located southwest of the Los Angeles Metropolitan area. www.maps.google.com.
Three Opuntia species native to Santa Catalina Island, California were studied in this research: Opuntia littoralis (Engelm.) Cockerell (Figure 1.3), Opuntia ficus-indica (L.)
Mill. (Figure 1.4), and Opuntia prolifera Engelm (Figure 1.5). O. littoralis and O. ficus- indica are prickly pear cactus species with flat pads and segmented joints. O. prolifera is a cyclindropuntia (tree-like cactus) with narrow, cyclindrical pads and segmented joints.
O. littoralis, O. ficus-indica and O. prolifera are distributed on the coastal slopes of the
4
Pacific Ocean from Southern California to Northern Mexico (1). O. littoralis and O. prolifera are native to the coastal slopes of the California Channel Islands, including
Santa Catalina Island. These two cactus species are the preferred nesting sites for the coastal cactus wren (Campylorhynchus brunneicapillus)(Figure 1.5) (2). O. ficus-indica has hybridized throughout the Channel Islands and inland, coastal slopes of Southern
California (1). O. ficus-indica has been exported to suitable climes in Southern Europe and Malta where it is grown for its fruit, which is known as an Indian fig. O. littoralis
(coastal prickly pear cactus) grows up to one meter tall and spreads several meters wide.
It has flat, rounded stems or cladodes and in the spring presents yellow and pink waxy flowers that yield edible purple fruit. O. ficus-indica (mission prickly pear cactus) is a tree-like cactus species reaching 4-5 m in height. O. ficus-indica has branched, flat pads.
It produces yellow or orange flowers that mature into juicy yellow, orange or purples fruits. O. prolifera (coastal cholla cactus) is sometimes classified within the subgenus
Cylindropuntia and reaches heights of up to 3 m. O. prolifera has a central trunk with branching, rounded tubercles. O. prolifera presents pink-magenta flowers in the spring that develop into edible fruits.
5
Figure 1.2. Photograph of coastal cactus wren (Campylorhynchus brunneicapillus) perched on cholla cactus. Reprinted from www.wikipedia.org.
Opuntia were eaten by humans. Fruit from cactus was an important food source for the Seri people of Northwestern Mexico (3). The Seri Indians consumed ripe Opuntia fruits, which they called “tunas”, raw (3). Fruit was dried and stored for later consumption. Seeds from the dry fruit were roasted before being consumed (3).
Additionally, the seeds were dried in the sun and then ground into a meal for cooking (3).
During the 1960’s, it was reported that a large number of Native Americans in several states of Mexico were living almost entirely off the tunas of Opuntia for at least four months of the year (4). These peoples also used the tunas to produced ‘queso de tuna’, a
6 flat cake that looks like cheese (4). This “cheese” contains 11% water and more than
70% sugar (4). The stem segments of the Opuntia cactus are called cladodes or pads, and the young pads of Opuntia are used as a vegetable in the southwestern United States and throughout Mexico (3). O. ficus-indica is the primary Opuntia species consumed.
Opuntia are still utilized to feed livestock; the thorns are removed so that they can be consumed safely by cattle. In Mexico, donkeys are chiefly fed the shoots of
Cylindropuntia (4).
Native Americans of both the United States and Mexico used the seeds of
Opuntia to produce red dye to dye clothing (3). Additionally, Opuntia are a food source for the cochineal insect, which produce a deep red dye. Aztec emperors, including
Montezuma, wore royal robes dyed by the dye harvested from the cochineal (3). Three of the six plants of O. ficus-indica used for this study contained active insects, which produced a cottony web on the exterior of the cactus pads.
The flat, fleshy pads of O. littoralis and O. ficus-indica were split and used by
Native Americans to bind wounds and burns (3). The Chumash people used O. littoralis as a poultice for wounds (5). O. ficus-indica is a popular herbal medicine in Mexico where it is used for diabetes, hypercholesterolemia, obesity, alcohol-induced hangover, colitis, diarrhea, benign prostatic hypertrophy (BPH), and atherosclerosis (6). Extracts of
O. ficus-indica have also been used to treat ulcers (7). The pharmacological activities of
O. ficus-indica have been reviewed (8). Although there is a rich ethnobotanical history of
Opuntia use, there has been only limited study of the volatiles of these plants. The floral volatiles of O. ficus-indica from Italy (9) and from Tunisia (10), and the fruit essential
7 oils of cultivated O. ficus-indica from Catania, Italy (11) and Sicily (12) have been reported.
Figure 1.3. Opuntia littoralis (Engelm.) Cockerell, which is commonly referred to as coastal prickly pear cactus. Photograph reprinted from www.wikipedia.org.
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Figure 2. O. ficus-indica L. (Mill).
Figure 1.4. Opuntia ficus-indica L. (Mill). which Figure 1.5. Opuntia prolifera Engelm., which is is commonly referred to as mission cactus. commonly referred to as coastal cholla. Photograph Photograph reprinted from the Catalina Island reprinted from the Catalina Island Conservancy. Conservancy. www.catalinaconservancy.org. www.catalinaconservancy.org.
1.3 Arizona-Sonora Desert Museum Opuntia Species
The Sonora Desert, which is considered to be a subtropical desert, is located in southeastern California, southwestern Arizona, and northern Mexico. Although the
Sonora Desert is one of the hottest and driest deserts in North America, it has a large diversity of species, and is home to at least 60 species of mammals, more than 350 bird species, 20 amphibians, 100 reptiles, approximately 30 species of native fish, and more than 2,000 species of plants (13). The portion of the Sonora Desert lying within the
United States is topographically divided into a lower, western section that includes the
Organ Pipe Cactus National Monument, and a higher, wetter eastern section that includes
Tucson and the Arizona-Sonora Desert Museum (Figure 1.6) (14). The scarcity and uncertain distribution of rainfall within the Sonora Desert is the single most important
9 climatic factor influencing the survival and distribution of plant species (15). The average rainfall for Tucson, Arizona and Ajo, Arizona is 318 and 213 mm per year, respectively (16).
Figure 1.6. Photograph of the Arizona-Sonora Desert Museum located near Tucson, Arizona. Reprinted from www.wikipedia.org.
The Sonora Desert lies in the Basin and Range geologic area wherein desert and grassland valleys are rimmed by parallel mountains extending north from Mexico (17).
These mountains are believed to be formed by active volcanoes 20-40 million years ago.
The Sonora Desert has differing soils that affect vegetation. Soil composition in the desert basins may be gravelly, sandy, or made up of clays, which may be humus poor and
10 gray in color or rich in lime and reddish in color (18). Soil of the Organ Pipe Cactus
National Monument is rich in lime (19). Soil at the Arizona-Sonora Desert Museum is rich with a unique mineral deposit called caliche that forms within granite rocks and soils
(18). Caliche is a reddish-brown layer of deposit formed when mineral-rich water flows through soil (18). The water leaves behind a calcium carbonate precipitate that hardens as it forms layers within the soil (18). Atmospheric calcium contained in dust and precipitation is shown to contribute to the formation of caliche soil (20).
This study investigates whether there are differences in the essential oil composition of two cactus species commonly found in the Sonora Desert, Opuntia acanthocarpa var. major (Engelm. & J.M. Bigelow) L.D. Benson (Figure 1.4) and
Opuntia phaeacantha var. discata (Griffiths) L.D. Benson & Walk. (Figure 1.5). These two species grow in both the lime-based soil of the Organ Pipe Cactus National
Monument and the caliche soil found at the Arizona-Sonora Desert Museum. O. acanthocarpa is a cylindro-opuntia native to the sandy soils of flats and washes in the desert at 300 to 1,000 m elevation from Sonora, Mexico, to Riverside County, California, and to the southern Counties of Arizona (21), including both the Organ Pipe Cactus
National Monument and the Arizona-Sonora Desert Museum. O. acanthocarpa var. major (commonly known as buckhorn cholla) is a shrub, arborescent plant, or a small tree with a trunk of 10 to 15 cm in diameter and a height of 1 to 2 m (21). The 2- to 5-cm tubercles are presented on joints of 15 to 30 cm long (21). The Pima Indians of Southern
Arizona steamed the flower buds in pits and utilized them for food (21). O. phaeacantha var. discata (syn. Opuntia engelmannii var. engelmannii) is a prickly pear cactus distributed in sandy soils of plains and grasslands at 500 up to 1500 m elevation (21).
11
The range of O. phaeacantha var. discata extends from Sonora and Chihuahua, Mexico through the California mountains bordering the deserts eastward to Utah and Texas (21).
The flat, jointed pads of O. phaeacantha var. discata are orbiculate to elliptic with a length of 20 to 40 cm long and 18 to 23 cm wide (21). In Mexico, cattle, deer, and other animals consume O. phaeacantha var. discata as forage (3). In fact, opuntias such as O. phaeacantha var. discata can be the principal component of a desert animal’s diet.
White-tail deer have been found to have a diet composed of 21% opuntia fodder (3). The native O’odham people utilized prickly pear cactus pads as a food source (18).
Additionally, the O’odham split and heated the cactus pads and applied to joints of the body to treat arthritis and rheumatism(18).
Samples for this study collected from the Organ Pipe Cactus National Monument were obtained from very gravelly, limy soil (19). The surface layer is light brown, very gravelly loam about 5 cm thick (19). The subsoil is pink, very gravelly loam about 20 cm thick and the substratum is white and pinkish gray, very gravelly loam and weakly cemented with lime to 1.5 m and more (19). This soil is composed of younger intermediate alluvial fan and terrace deposits of poorly sorted cobbles, pebbles, and sand, with lesser amounts of silt, clay and boulders (22). The Arizona-Sonora Desert Museum is located in the Tuscon Mountain range located in Tucson, Arizona. Soil in the Tucson
Mountain range lacks argillic horizons and has weak structural development characterized by moderately to highly cemented calcic horizons at depths in excess of 20-
25 cm (23). Petrocalcic horizons are found throughout the upper 10 m of alluvium comprising the land surface within the Tucson Mountain range (23).
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Figure 1.7. O. acanthocarpa var. major (Engelm.. Figure 1.8. O. phaeacantha var. discata (Griffiths) & J.M. L.D. Bigelow) L.D. Benson growing in the Benson & Walk.growing at the Organ Pipe Cactus Sonoran Desert of Southern Arizona. Photograph National Monument. Photograph reprinted from reprinted from www.wikipedia.org. www.wikipedia.org.
1.4 Stenocereus thurberi Engelm. Growing at Organ Pipe Cactus National Monument Stenocereus thurberi Engelm. (Figure 1.6), also known as organ pipe cactus, is a columnar cactus native to the Sonora Desert, which is located in Southwestern Arizona reaching into Northern Mexico. In Arizona organ pipe cactus is only found in Organ
Pipe Cactus National Monument and the nearby Tohono O’odham Indian Reservation.
In Mexico the organ pipe cactus is found in Sonora and Southern Baja California. S. thurberi grows 3 meters or more in height with the outside perimeter of the columns reaching a width of 4 meters or more (21). The cactus blooms white or lavender from late spring to early summer and produces an edible fruit in late Summer. These bloom at night and are pollinated by bats. Each mature column contains a porous wooden skeleton approximately 5 cm diameter that runs the length of each column.
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S. thurberi fruit, which is called “pitahayas”, is a favorite of the Seri Indians of
Northwestern Mexico. The Seri Indians consumed pitahayas and used them to make wine (3). Additionally, the Seri Indians utilized S. thurberi as a medicine to cure aches and pains (3). Arms of organ pipe cactus were used in Indian dwellings and to cactus extracts were used to make boat sealant (24).
Figure 1.9. Stenocereus thurberi Engelm. growing in the Sonora Desert. Reprinted from www.wikipedia.org.
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1.5 Necrotic Opuntia littoralis (Engelm.) Cockerell and Stenocereus thurberi Engelm.
Drosophila is a model organism for the study of ecology, genomics, and evolutionary biology. There are more than 2,000 identifiable species of Drosophila (25) that are widely distributed. Drosophila species have been identified that are generalists adapted to oviposit in several hosts (25). Other Drosophila species have been identified that are limited to feeding and ovipositing on a single host (25). Drosophila are known that utilize fruits, flowers, soil, land crabs, spider eggs, and cacti as hosts (25). The adaptation of Drosophila to numerous hosts has led to the specialization and speciation of
Drosophila. This variety of hosts affects the reproductive biology of Drosophila and, ultimately, the evolution of Drosophila (25). The genome of twelve species of
Drosophila has been mapped (25). Coupling the knowledge of Drosophila genomics with host ecology allows the study of evolutionary processes (25).
Drosophila melanogaster, the fruit fly, is a model system for investigating human disease because more than 75% of human diseases have a homologue in flies (26). D. melanogaster is a generalist that utilizes fruit, flowers, other plant parts, and green cactus as a food source and to oviposit young (25). Drosophila mojavensis is a cactophilic fly that utilizes the necrotic rots of cactus as its host. D. mojavensis populates areas of both
Santa Catalina Island, Califoronia, the Mojave Desert of Southern California and Western
Arizona, the Sonoran Desert of Southwestern Arizona, and the desert areas of Sonora
Mexico, and Baja California, Mexico (27). A subspecies of D. mojavensis is geographically limited to Santa Catalina Island, California and utilizes necrotic rots of O.
15 littoralis (27). A second subspecies of D. mojavensis is geographically limited to the
Sonora Desert and utilizes necrotic rots of S. thurberi as a food source and a site to oviposit (27) except for the Desemboque, Mexico region where agria cactus is utilized
(Stenocereus gummosus)(28).
Although the necrotic rots of these cactus species are utilized by D. mojavensis, these rots are lethal when consumed by D. melanogster causing 100% mortality upon 48 hours of exposure. This study identified the components of the essential oil of the necrotic rots of both O. littoralis and S. thurberi. Compounds identified in the essential oil were investigated to determine the degree of toxicity to D. melanogaster. The identification of compounds in the cactus rots that are toxic to fruit flies may aid in understanding the biochemical processes evolved in the detoxification of these compounds. Because detoxifying enzymes of D. melanogaster are similar to detoxifying enzymes of humans, understanding the biochemical processes and evolutionary biology of these processes may lead to a better understanding of human detoxifying enzymes.
The volatiles of the cactus rot attract D. mojavensis for feeding and breeding (29).
The varying chemistry of the two different cactus species provides substrates for different species of Drosophila. It is suggested that chemosensory perception is a target of natural selection leading to ecological specialization and divergence of species (30). Ecological specialization may lead to genetic diversity within a species, which may lead to speciation (31) Thus, the chemistry of both O. littoralis and S. thurberi are part of the complex interplay of ecological specialization, evolutionary biology, genetic diversity, and genomics in the Drosophila model. This study investigates the volatile fraction of
16 the headspace surrounding the cactus rots to ascertain what volatile compounds may be attracting the flies to the rot.
1.6 Silica Bodies in Cactus
Silica, SiO2, has been identified in the skin of certain columnar cacti (24). Silica bodies have been identified in all species of Stenocereus (24). The silica bodies that have been identified are starburst-shaped crystals (32). Identification was made based on hydrofluoric acid testing (32). This testing was basesd on the fact that the hydrofluoric acid is the only acid that will react with silica. To date, silica bodies have not been found in any cactus species other than the Mexican columnar cacti. There is no report of silica bodies being discovered in any of the Opuntia species. Additionally, the silica bodies previously identified have only been found in the skin or epidermis of the columnar cacti.
Although silica bodies have been identified in more than 20 plants, including rice, bananas, and horsetails, no organism has been identified that can store silicon in a form other than SiO2 (24).
Silicon is found in soils throughout the world in the form of silicic acid [Si(OH)4
- or Si(OH)3O ] at a dry weight concentration of less than 1% to more than 45% (33).
Silicic acid is absorbed via the plant roots and is transported via silicon transporters, polymerized into silica bodies, and stored in both cell walls and vacuoles (34). Silicon is found to be beneficial for the following important crops: rice (35), wheat (36), oat (37), cucumber (38), and tomato (39). Plants grown in medium lacking silicon may suffer structural, growth, and reproduction abnormalities (40). Metal toxicity, salinity, drought
17 and temperature stress are found to be alleviated by the addition of silicic acid to the soil
(40). These beneficial effects have lead to the incorporation of silicon into fertilizers.
How silicic acid exerts these beneficial effects is unknown.
Silicon can be methylated and phenylated commercially via chlorine and metal catalysts such as copper, zinc, and platinum. No organism has been identified that can methylate or phenylate silicon. Methylated and phenylated siloxanes are referred to as oligodimethylsiloxanes and polydimethylsiloxanes (PDMS). Polydimethylsiloxanes have the following formula: Me3SiO (SiMe2O)n SiMe3 where n = 0, 1, ...
Polydimethylsiloxanes can be linear or cyclic. The cyclic forms are commonly used in the commercial production of skin care products. The cyclic forms are also a contaminant in the production of linear oligosiloxanes. Linear oligosiloxanes have extremely high melting points, and cyclic oligosiloxanes have low boiling points.
18
CHAPTER II
Materials and Methods
This chapter discusses the laboratory procedures used in in this dissertation. This chapter introduces hydrodistillation and dynamic headspace extraction procedures and the analytical procedures of gas chromatography-mass spectrometry and nuclear magnetic resonance. Compounds identified in the necrotic rots were subjected to toxicity experiments to establish LC50 toxicity levels for fruit flies. Fruit flies were subjected to these compounds to determine whether they had an olfactory preference for any of the compounds. Additionally, microscopy methods for the compound and confocal microscopy are discussed.
2.1 Plant Collection and Extraction
This section discusses the amount of cactus specimen used in each extraction procedure, how each extraction was performed, and the amount of extract recovered.
Because cyclic siloxanes are common to over-the-counter skin products, hands were
19 washed thoroughly prior to handling any specimens. Additionally, specimens did not come into direct contact with the investigator’s hands. All specimens were handled with clean instruments.
2.1.1 Plant Collection and Extraction of Santa Catalina Island California Species
Specimens of green cactus were identified and collected from Santa Catalina
Island, California by Ben Coleman, of the Catalina Island Conservancy. Three green pads of six different plants of each species of O. littoralis and O. ficus-indica were analyzed. The green pads of both cacti were chopped and hydrodistilled using a Likens-
Nickerson apparatus (Figure 1.1). Continuous extraction of the distillate with chloroform for four hours gave a dark yellow-brown extract for both species of cactus, which was stored at 4C until analysis. Similarly, 156.51 g of O. prolifera was hydrodistilled to give
349.8 mg essential oil. The extract was maintained at 4C until analyzed.
20
Figure 2.1. Likens-Nickerson apparatus used for hydrodistillation.
2.1.2 Collection and Extraction of Arizona-Sonora Desert Museum Species
Cactus specimens were identified and collected from the Organ Pipe Cactus
National Monument by Tim Tibbits of the National Park Service, U.S. Department of the
Interior. Specimens of cactus were also collected by Julie Wiens of the Arizona-Sonora
Desert Museum. Samples were chopped and frozen at -20C until studied. The plant samples were hydrodistilled using a Likens-Nickerson apparatus with continuous
21 extraction with chloroform for a four-hour period. The chloroform was evaporated from the distillates to give the essential oils. The distillates were stored at 4C until analysis.
2.1.3 Collection and Extraction of Stenocereus thurberi Engelm.
Green S. thurberi was identified and collected by Luciano Matzkin. The green organ pipe cactus was pureed and stored at -20C. The plant sample (48.69 g) was hydrodistilled using a Likens-Nickerson apparatus with continuous extraction with chlorofo rm for a four-hour period. The chloroform was evaporated from the distillates to give the essential oil (52.1 mg). The distillate was stored at 4C until analysis.
2.1.4 Collection and Dynamic Headspace Extraction of Necrotic and Lab Specimens of Opuntia littoralis (Engelm.) Cockerell and Stenocereus thurberi Engelm.
Necrotic specimens (1.47 kg) of O. littoralis were identified and collected from
Santa Catalina Island, California by Ben Coleman, of the Catalina Island Conservancy.
Differing states of necroses were identified. Specimens were chopped and allowed to rot for 3 weeks at 25C until subjected to dynamic headspace extraction. The rot had a sweet, pleasant smell. Necrotic specimens (3.42 kg) of Stenocereus thurberi Engelm. were identified and collected by Tim Tibbits of the Organ Pipe Cactus National
Monument, National Park Service. The dynamic headspace extraction was performed by placing necrotic specimens in a Fischer Scientific vacuum oven connected to a GAST power-operated air pump (Figure 1.2). The vacuum temperature was maintained at 38C
22 and air was pumped across the sample and through a vacuum flask that was placed in an insulated vacuum flask holder lined with dry ice for 6 hrs. The volatiles accumulated in ice within the vacuum flask. The ice was melted at 25C and saturated with NaCl.
Dichloromethane (DCM) was applied in an amount equal to the ice extract volume and shaken vigorously for 2 minutes. The extract was allowed to partition and the DCM extract was removed. This step was repeated 3 times. The oil was stored at 4C until analysis.
Figure 2.2. Photograph of apparatus for dynamic headspace extraction. Note specimen placed on top of vacuum oven. Vacuum flask to the right of vacuum oven was placed in silver flask holder, which was lined with dry ice.
23
810.45 g of O. littoralis extracted via dynamic headspace produced 102 mg of a clear colorless extract. The S. thurberi dynamic headspace extraction produced 156.9 mg of dark yellow distillate. The oil fractions were stored at 4°C until further analysis was stored at 25C until analysis.
After dynamic headspace extraction, necrotic specimens were hydrodistilled using a Likens-Nickerson apparatus. Continuous extraction of the distillate with chloroform for four hours gave a clear colorless oil with a yield of 1.1 mg O. littoralis essential oil and
880.7 mg of dark yellow, brown S. thurberi essential oil. The S. thurberi oil had a strong, offensive odor. Both oils were stored at 4C until analysis.
Lab-created rots were provided by Luciano Matzkin. The lab rots were prepared by incubating autoclaved cactus for 5 weeks at 25C with the following microbes that were previously identified in native rots: Pectobacterium cacticada, Pichia cactophila,
Pichia amenthionina, Candida sonorensis, Candida ingens, and Sporopachydermia cereana.
O. littoralis lab-created rot exuded a strong, offensive odor. The artificially-rotted cactus (48.30 grams) was blended and stored at -20C until hydrodistilled with a Likens-
Nickerson apparatus. Continuous extraction of the distillates with chloroform for four hours gave a slightly yellow translucent distillate. The extraction procedure produced a yield of 1.1 mg, which was stored at 4C until analysis.
The S. thurberi lab rot exuded a strong, offensive odor. The artificially-rotted cactus (43.63 grams) produced a yield of 1.1 mg with a yellow-green color. The oil was stored at 4C until analysis.
24
2.2 Microscopic Investigation
Unstained samples of O. littoralis, S. thurberi, and O. ficus-indica were sliced thinly and viewed with a Nikon E600 compound microscope with a Q Imaging QICAM
Fast 1394 camera attached. Samples of O. littoralis, S. thurberi, and O. ficus-indica were also viewed with a Zeiss LSM700 confocal microscope. Cactus specimens were thinly sliced and stained with PDMPO, (2-(4-pyridyl)-5-((4-(2- dimethylaminoethylaminocarbamoyl) methoxy)-phenyl) oxazole. PDMPO is a fluorescent probe that reacts with silica under acidic conditions causing the silica to fluoresce, which can be observed with a microscope. PDMPO does not react with silicic acid (41), therefore, silica presence can be confirmed. The specimens stained with
PDMPO were placed in the dark for 5 days at 25C before analysis.
2.3 Chromatographic Identification of Compounds
The cactus essential oils were subjected to gas chromatographic-mass spectral analysis on an Agilent system consisting of a Model 6890 gas chromatograph, a Model
5973 mass selective detector [MSD, operated in EI mode (electron energy=70ev), scan range=40-400amu, and scan rate = 3.99 scains/sec], and an HP-5ms fused silica capillary with a (5% phenyl)-poly-methylsiloxane stationary phase, film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. inlet temperature was
200ºC and interface temperature was 280ºC. The GC oven temperature program was used as follows: 60ºC initial temperature, hold for 5mins; increased at 3ºC/min to 280ºC;
25 increased at 3ºC/min. A 1% w/v solution of the sample in CH2Cl2 was prepared and injected using a 10:1 split ratio.
Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature (42) and stored on the MS library [NIST database (G1036A, revision D.01.00)/ChemStation data system
(G1701CA, version C.00.01.080)]. A minimum of 95% confidence on the NIST database was attained prior to the identification of any compound. The percentages of each component are reported as raw percentages based on total ion current without standardization. The compositions of the cactus essential oils listed in all tables within this dissertation except Table 3-14 do not include the cyclic siloxane components. Cyclic siloxanes are not listed because they are not essential oils and because it can not be confirmed that they are actual components of the cacti studied in this dissertation.
2.4 Carboxylic Acid Esterification
To determine whether the broad carboxylic acid peaks seen in the GC-MS spectra of the lab-rot specimens were obscuring other compounds within the extract that had close retention times, the carboxylic acids were derivatized to methyl esters (FAME) prior to analysis by gas chromatography-mass spectrometry. A sample of 0.3 mg of necrotic extract was added to 150 µL acetylchloride in 5% methanol (freshly prepared) and mixed at 4,000 rpm for 10 seconds. The sample was heated at 75C for 10 minutes and mixed at 4,000 rpm for 10 seconds three times. Bicarbonate was added and the
26 sample mixed at 4,000 rpm for 30 seconds. The sample was allowed to sit for 2 minutes before the extract was collected from the top layer.
2.5 Fly Toxicity Testing
Drosophila melanogaster flies were tested with the following compounds: p- cresol, p- methyl anisole, phenyl ethyl alcohol, 2-heptanone, oleic acid, butanoic acid, benzaldehyde, pentanal, nonanoic acid, hexanoic acid, octanoic acid, hexadecane, 2- nonanone, isophorone, phenol, palmitic acid, and green and necrotic specimens of O. littoralis and S. thurberi. Reference compounds were obtained from Sigma-Aldrich at the highest grade of purity available. Three replicates of ten one-day-old flies-5 male and
5 female-were tested upon 24 hrs of exposure to media containing each compound.
Media was prepared by mixing Carolina Biological Supply fly media with a 1% solution of each compound in DMSO. The percentage of each compound was increased until
100% fly mortality was reached upon 24 hrs of exposure, or until the maximum solubility of the tested compound was reached. The Reed-Muench method was used to determine the LC50 for each compound tested.
2.6 Drosophila melanogaster Olfactory Preference Test
A clear plexiglass box was constructed so that vials of all tested compounds and cactus specimens were suspended from a shelf in the box. The flies were able to enter the vial containing the compound being tested (2%) or cactus specimen of their choosing.
27
The number of flies in each vial was counted at two hour time intervals. Three replicates were performed which tested a total of approximately 1,800 flies.
2.7 Structure Determination
Nuclear magnetic resonance (NMR) was used to determine the structures of the white particulate isolated from both O. littoralis and S. thurberi. 15 mg of white particulate was added to 2 mL of CDCl3 and vortexed for 15 minutes prior to analysis.
The following NMR spectra were obtained : proton (1H), and carbon (13C). 1D NMR data were obtained on a 500 MHz Varian INOVA spectrometer at 25°C. The NMR data were processed and analyzed using Mnova NMR software. The solvent used to dissolve the samples (CDCl3) was also used as references in the spectra.
2.8 Single-Crystal X-Ray Diffraction
Crystals were dissected from O. littoralis and S. thurberi and sent to the
University of Alabama, Tuscaloosa for small crystal x-ray diffraction analysis. Dr.
Patrick Berber, Department of Chemistry, Center for Green Manufacturing, analyzed both samples on a Bruker SMART diffractometer with an Apex II detector.
28
CHAPTER III
RESULTS
This chapter gives the results of the experiments conducted for this dissertation.
Each of the cactus specimens contained very little oil. The oil compositions of each species was complex and varied. O. littoralis contained the most complex oil which was composed of 106 different identifiable compounds. None of the Opunita species studied shared any major components in similar percentages. The oil composition of the two
Opuntia species growing in different locations within the Sonora desert had vast different oil content.
Although the lab-rotted specimens of both O. littoralis and S. thurberi had similar essential oil composition, the oil content of the field rot specimens varied significantly from the lab rots. The lab rots were dominated by carboxylic acids while the field rot specimen of S. thurberi was dominated by p-cresol. Several compounds contained within the rots were highly toxic to D. melanogaster including p-cresol, nonanoic acid, phenol, and octanoic acid. The fruit flies lacked any preference for the compounds identified within the cactus rots.
29
Silica crystals were identified in O. littoralis, O. ficus-indica, and S. thurberi.
The crystal bodies within the Opuntia species were starbursts while the crystals in S. thurberi were rods. Large, cyclic oligosiloxanes were identified in the GC-MS spectra of
O. littoralis, O. ficus-indica, and S. thurberi. One plant of O. littoralis was identified that lacked both silica crystals and cyclic siloxane. This suggests that the presence of silica crystals and cyclic siloxane are related. The chemistry that would enable cyclic siloxanes to be formed within the cactus species studied in this dissertation is unknown.
3.1 Compounds Identified in Santa Catalina Island, California Opuntia Species
The essential oils of O. littoralis, O. ficus-indica, and O. prolifera were obtained in low yields: 0.28%, 0.0012%, and 0.22%, respectively. A total of 106 compounds were identified in O. littoralis, representing 94.5 % of the composition (Table 3.1).
Sixty-five compounds were identified in O. ficus-indica, representing 96.5 % of the oil composition (Table 3.2). Sixty-two compounds were identified in O. prolifera, which accounts for 94.7% of the oil composition (Table 3.3).
O. littoralis oil was made up of 47.2% terpenoid-derived compounds, 29.1% fatty-acid-derived compounds, 7.6% alkanes, and 10.6% furanoid and benzenoid compounds. The compounds of greatest concentration in O. littoralis were cis-linalool oxide (10.8%), trans-linalool oxide (8.8%), palmitic acid (4.4%), 2-furaldehyde (3.8%),
2-hexanol (3.5%), 7,10-epoxy-2,6,10-trimethyl-2,5,11-dodecatriene (3.4%), nonanal
(3.4%), and lauric acid (2.7%). O. littoralis oil also contained small amounts of santene
(0.3%), vitispirane (1.0%), and (9Z)-tricosene (0.8%). Santene is an unsaturated terpene
30 found in East Indian sandalwood oil (43) and black spruce needles (44). Vitispirane is a norisoprenoid found in grapes and wine (45). (9Z)-Tricosene (muscalure) is a sex pheromone produced by female common houseflies, Muscadomestica to attract males
(46). The high concentrations of furanoid linalool oxides in O. littoralis cladode essential oil was is unexpected. Linalool oxides are important fragrance components of floral (47)
(48) (49) (50) and fruit (51) (52) volatiles, including Cereus peruvianus (Cactaceae) fruit
(53) (54), but their presence in cactus pads seems unusual.
Fatty acids dominated the essential oil of O. ficus-indica pads with linoleic acid
(22.3%), palimitic acid (12.7%), lauric acid (10.5%), and myristic acid (4.2%) as the most abundant. In addition to fatty acids, there were also notable quantities of (E)-phytol
(8.0%) and (9Z)-tricosene (muscalure, 6.7%). And p-vinylguaiacol (3.4%). In contrast,
O. ficus-indica fruit grown in Catania, Italy was found to be primarily composed of (2E)- hexen-1-ol (54.7-58.0%) and 1-hexanol (25.2-29.5%) (11). Neither of these alcohols was detected in the O. ficus-indica cladode oil in this current study. The fruit volatiles of a cultivar of O. ficus-indica growing in Sicily did contain large quantities of palmitic acid
(33.0%) and fatty acid esters (14.0%) (12). Fatty acids have been found to be relatively abundant in the floral essential oils of O. ficus-indica (10). Long-chain alkanes made up a significant portion (8.1%) of the O. ficus-indica cladode oil in this study. Large quantities of alkanes have been found in the skin volatiles from O. ficus-indica fruit (12) as well as O. ficus-indica flowers (9). O. ficus-indica cladode oil contained only small quantities of linalool and linalool oxides, in contrast to O. littoralis cladode oil (see above). (9Z)-Tricosene was found in larger quantities in O. ficus-indica oil than in O.
31 littoralis oil (see above). Interestingly, (9Z)-pentacosene (1.4%), which is a sex pherom one of several insect species (55) (56), was also found in O. ficus-indica oil.
O. prolifera oil was dominated by alkanes (47.1%), and the primary component was heptadecane (19.2%). In addition to normal alkanes, e.g., tetradecane (1.3%), pentadecane (5.1%), hexadecane (3.8%), etc., there were several branched alkanes present in O. prolifera oil, including norpristane (1.0%), pristane (1.4%), and phytane
(1.0%). Fatty aldehydes (18.4%), both saturated and unsaturated, were also common in
O. prolifera oil. Another notable component in O. prolifera oil was p-menth-4-en-3-one
(3.5%), which has been identified in Ficus (57) and Ephedra (58) species.
None of the three cactus plants examined in this work shared any major components in similar percentages, and there were only 16 compounds found in all three cactus species. Compounds common to all three species were trans-linalool oxide, geranyl acetone, (E)--ionone, pentadecane, lauric (dodecanoic) acid, heptadecane, myristic (tetradecanoic) acid, octadecane, nonadecane, palmitic (hexadecanoic) acid, heneicosane, (E)-phytol, docosane, hexacosane,heptacosane, and nonacosane.
Table 3.1. Essential oil components of Opuntia littoralis.
RIa RTb Compound %c
801 3.798 Hexanal 0.9
810 3.863 2-Hexanold 3.5
838 4.344 2-Furaldehyde 3.8
32
Table 3.1 (Continued)
855 4.756 (2E)-Hexenal 0.5
858 4.817 (3Z)-Hexenol 0.2
871 5.159 1-Hexanol 0.3
886 5.694 Santene 0.3
904 6.214 Heptanal 0.7
910 6.364 2-Acetylfuran 0.2
933 6.570 Prenyl acetatee 0.1
961 8.296 (2E)-Heptenal 0.4
964 8.462 Benzaldehyde 0.5
967 8.597 Methylfurfural 0.4
979 9.126 -Pinene tre
982 9.270 1-Octen-3-ol 0.2
989 9.612 6-Methyl-5-hepten-2-one 0.4
993 9.799 2-Pentylfuran 0.6
998 10.033 (2E,4Z)-Heptadienald 0.1
1004 10.303 Octanal 0.4
1011 10.645 (2E,4E)-Heptadienal 0.3
1025 11.264 p-Cymene 1.4
1028 11.432 Limonene 0.1
1031 11.547 1,8-Cineole 0.6
1044 12.175 Phenylacetaldehyde 1.4
33
Table 3.1 (Continued)
1049 12.397 (E)--Ocimene 0.2
1059 12.856 (2E)-Octenal 0.4
1067 13.228 Acetophenone 0.7
1074 13.497 cis-Linalool oxide (furanoid) 10.8
1090 14.232 trans-Linalool oxide (furanoid) 8.8
1096 14.611 Methyl benzoate 0.4
1102 14.827 Linalool 1.7
1107 15.034 Nonanal 3.4
1117 15.616 α-Cyclocitrale 0.3
1126 16.052 α-Campholenal 0.4
1126 16.171 1-Phenyl-2-propanone 0.2
1140 16.695 (E,E)-2,6-Dimethyl-1,3,5,7-octatetraene (= Cosmene)e 0.2
1143 16.873 Camphor 0.2
1150 17.181 1,4-Dimethylcyclohex-3-enyl methyl ketonee 0.4
1154 17.372 (2E,6Z)-Nonadienal 0.4
1160 17.688 (2E)-Nonenal 0.6
1171 18.194 Ethyl benzoate 0.2
1177 18.472 p-Mentha-1,8-dien-4-ole 0.6
1180 18.615 Naphthalene 0.1
1184 18.805 p-Methylacetophenone trd
1187 18.948 p-Cymen-8-ol 1.4
1192 19.087 a-Terpineol 2.1
34
Table 3.1 (Continued)
1194 19.363 Methyl salicylate 0.6
1195 19.467 g-Terpineol 0.9
1206 19.880 Decanal 0.7
1214 20.039 p-Menth-1-en-9-ale,g 0.6
1216 20.405 p-Menth-1-en-9-ale,g 0.6
1219 20.512 -Cyclocitral 0.6
1227 20.835 cis-Sabinene hydrate acetate 0.6
1256 22.196 Geraniol 0.5
1262 22.434 (2E)-Decenal 0.2
1278 23.171 Vitispiranee 1.0
1293 23.890 (2E,4Z)-Decadienal 0.3
1298 24.166 Tridecane 0.1
1303 24.497 Undecanal 0.4
1315 24.902 (2E,4E)-Decadienal 0.8
1364 26.961 (2E)-Undecenal 0.2
1375 27.450 α-Copaene 0.9
1384 27.582 Decanoic acid 0.4
1384 27.845 (E)--Damascenone 0.3
1400 28.524 Tetradecane 0.4
1454 30.745 Geranyl acetone 0.6
1460 30.995 Alloaromadendrene 0.4
35
Table 3.1 (Continued)
1465 31.183 (2E)-Dodecenal 0.3
1478 31.673 g-Muurolene 0.3
1488 32.091 (E)-b-Ionone 0.9
1500 32.697 Pentadecane 1.9
32.780 (3S,6R,7E)-7,10-Epoxy-2,6,10-trimethyl-2,5,11- 1503 1.4 dodecatrienee,h
33.194 (3S,6S,7E)-7,10-Epoxy-2,6,10-trimethyl-2,5,11- 1515 3.4 dodecatriene,e,h
1525 33.684 δ-Cadinene 0.2
1578 35.600 Lauric acid 2.7
1581 35.775 (E,E)-4,8,12-Trimethyl-1,3,7,11-tridecatetraenee 1.5
1600 36.493 Hexadecane 0.7
1614 36.553 Tetradecanal 0.4
1644 38.093 τ-Muurolol 0.2
1657 38.737 α-Cadinol 0.4
1669 39.068 (2E)-Tetradecenal 0.2
1674 39.301 Cadalene 0.5
1700 40.250 Heptadecane 0.7
1764 42.546 Benzyl benzoate 1.4
1771 42.816 Myristic acid 0.7
1800 43.761 Octadecane 0.3
1871 45.279 Pentadecanoic acid 0.8
1886 46.456 n-Hexadecanol 0.5
36
Table 3.1 (Continued)
1900 47.270 Nonadecane 0.4
1968 49.713 Palmitic acid 4.4
2000 50.321 Eicosane 0.2
2033 51.255 (E,E)-Geranyl linalool 0.4
2100 53.389 Heneicosane 0.4
2113 53.872 (E)-Phytol 1.2
2142 54.820 Linoleic acid 2.2
2167 55.229 Ethyl lineolate 0.4
2200 56.318 Docosane 0.1
2279 58.385 (9Z)-Tricosene (= Muscalure) 0.8
2300 59.137 Tricosane 0.5
2400 61.845 Tetracosane 1.1
2500 65.661 Pentacosane 0.3
2600 66.931 Hexacosane 0.1
2700 69.342 Heptacosane 0.1
2800 71.672 Octacosane 0.1
2830 72.329 Squalene 0.2
2900 73.930 Nonacosane 0.1
37
Table 3.1 (Continued)
3000 76.093 Triacontane 0.1
Total Identified 94.5 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. cThe percentages of each component are reported as raw percentages based on total ion current without standardization. dAbundant components are indicated in bold. e Identification is tentative and based on mass spectrum only. ftr = “trace” (< 0.05%). f Correct stereoisomer not determined. gStereochemistry may be reversed.
Table 3.2. Essential oil components of Opuntia ficus-indica.
RIa RT b Compound %c
986 13.150 Hexanoic acid trc
1072 13.498 cis-Linalool oxide (furanoid) tr
1083 13.987 Heptanoic acid tr
1088 14.248 trans-Linalool oxide (furanoid) 0.1
1100 14.827 Linalool Tr
1169 18.086 cis-Linalool oxide (pyranoid) 0.1
1174 18.339 trans-Linalool oxide (pyranoid) Tr
1183 18.748 Octanoic acid Tr
1185 18.865 p-Cymen-8-ol 0.3
1190 19.110 α-Terpineol 0.3
1205 19.850 Decanal tr
1221 20.615 p-Vinylphenol 0.2
38
Table 3.2 (Continued)
1250 22.146 Geraniol 0.1
1277 23.156 Vitispiranee 0.1
1279 23.244 Nonanoic acid 0.6
1294 23.906 Thymol 0.1
1313 24.871 p-Vinylguaiacol 3.4
1356 26.648 Benzalacetonee 0.1
1375 27.278 Decanoic acid 0.8
1454 30.727 Geranyl acetone 0.2
1462 31.060 Cerulignol (= 2-Methoxy-4-propylphenol)e 1.0
1477 31.677 Undecanoic acid 0.4
1487 32.072 (E)--Ionone 0.1
1500 32.645 Pentadecane 0.2
33.549 (3S,6S,7E)-7,10-Epoxy-2,6,10-trimethyl-2,5,11- 1513 0.2 dodecatrienee
1524 33.671 δ-Cadinene 0.2
1545 34.338 Nerolidol oxide isomere,f 1.0
1557 35.176 Nerolidol oxide isomere,f 0.4
1562 35.279 Nerolidol oxide isomere,f 0.4
1566 35.946 (E)-Nerolidol 0.2
1569 36.510 Lauric acidg 10.5
1614 37.034 Tetradecanal 0.2
1628 37.579 Megastigma-5,8(Z)-dien-4-onee 0.2
39
Table 3.2 (Continued)
1644 38.153 τ-Muurolol 0.2
1657 38.632 α-Bisabolol oxide B 0.4
1671 39.330 Tridecanoic acid 1.0
1700 40.236 Heptadecane 0.1
1762 42.489 Eupatoriochromene 1.0
1772 43.248 Myristic acid 4.2
1800 43.762 Octadecane 0.1
1844 44.691 Neophytadiene 0.2
1850 44.953 6,10,14-Trimethylpentadecan-2-one 0.6
1860 45.664 (10Z)-Pentadecenoic acid 1.9
1875 46.152 Pentadecanoic acid 1.6
1886 46.532 n-Hexadecanol 0.9
1900 47.124 Nonadecane 0.1
1922 47.752 (5E,9E)-Farnesyl acetone 0.3
1943 48.709 Isophytol 0.4
1948 48.862 Palmitoleoic acid 0.8
1968 50.009 Palmitic acid 12.7
2032 51.252 Isopropyl palmitate 0.7
2036 51.320 (E,E)-Geranyl linalool 0.7
2078 52.589 Octadecanol 0.9
2100 53.424 Heneicosane 0.8
2114 54.066 (E)-Phytol 8.0
40
Table 3.2 (Continued)
2139 54.500 Linoleic acid 22.3
2283 58.272 (9Z)-Tricosene (= Muscalure) 6.7
2300 59.242 Tricosane 1.6
2400 61.902 Tetracosane 0.8
2477 62.701 (9Z)-Pentacosene 1.4
2500 64.497 Pentacosane 1.3
2700 69.405 Heptacosane 1.2
2800 71.699 Octacosane 0.5
2830 72.340 Squalene 0.3
2900 73.965 Nonacosane 1.0
2997 76.043 Triacontenee,h 0.2
3000 76.114 Triacontane 0.2
Total Identified 96.5 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. cThe percentages of each component are reported as raw percentages based on total ion current without standardization. dtr = “trace” (< 0.05%). e Identification is tentative and based on mass spectrum only. f Correct stereoisomer not determined. gAbundant components are indicated in bold. h Correct regioisomer not determined.
41
Table 3.3. Essential oilcomponents of Opuntia prolifera.
RIa RTb Compound %c
857 4.804 (3Z)-Hexenol 0.7
870 5.144 1-Hexanol 1.3
903 6.203 Heptanal 0.2
933 7.010 Prenylacetated 0.2
1004 10.293 Octanal 0.3
1043 12.134 Phenylacetaldehyde 0.7
1058 12.828 (2E)-Octenal 0.1
1066 13.180 Acetophenone 0.2
1072 13.466 1-Octanol 1.3
1088 14.248 trans-Linalool oxide (furanoid) 0.1
1105 15.046 Nonanal 3.2
1151 17.350 (2E,6Z)-Nonadienal 0.6
1160 17.659 (2E)-Nonenal 0.9
1172 18.865 Nonanol 0.3
1205 19.110 Decanal 2.0
1219 20.470 -Cyclocitral 0.6
1246 21.727 p-Menth-4-en-3-oned 3.5
1261 22.421 (2E)-Decenal 1.7
1277 23.148 Vitispiraned 0.4
1280 23.246 Nonanoic acid 1.1
42
Table 3.3 (Continued)
1300 24.159 Tridecane 0.9
1303 24.479 Undecanal 0.5
1315 24.878 (2E,4E)-Decadienal 0.8
1363 26.949 (2E)-Undecenal 0.4
1379 27.499 Decanoic acid 1.6
1392 28.164 Undecanol 0.2
1400 28.512 Tetradecane 1.3
1410 28.903 Dodecanal 3.3
1454 30.718 Geranyl acetone 0.6
1463 31.090 2,6,10-Trimethyltridecane 0.9
1487 32.058 (E)--Ionone 0.5
1493 32.312 Dodecanol 0.3
1500 32.655 Pentadecane 5.1
1511 33.055 Tridecanal 0.6
1527 34.915 4-Methylpentadecane 0.4
1565 35.131 2-Methylpentadecane 2.2
1570 35.501 Dodecanoic acid 5.3
1600 36.490 Hexadecane 3.8
1614 37.015 Tetradecanal 2.4
1645 38.224 7-Methylhexadecane 0.3
1650 38.350 2,6,10-Trimethylpentadecane (= Norpristane) 1.0
1670 39.391 n-Tetradecanol 1.2
43
Table 3.3 (Continued)
1700 40.328 Heptadecanee 19.2
1707 40.469 2,6,10,14-Tetramethylpentadecane (= Pristane) 1.4
1713 40.760 Pentadecanal 0.7
1800 43.760 Octadecane 1.8
1808 44.075 2,6,10,14-Tetramethylhexadecane (= Phytane) 1.0
1815 44.312 Hexadecanal 0.5
1839 45.294 6,10,14-Trimethylpentadecan-2-one 2.0
1885 46.564 n-Hexadecanol 1.6
1900 47.116 Nonadecane 2.4
1962 49.292 Palmitic acid 2.7
2000 50.306 Eicosane 0.5
2029 51.117 Isopropyl palmitate 1.5
2090 53.192 Methyl lineolate 0.5
2100 53.382 Heneicosane 2.1
2112 53.858 (E)-Phytol 1.0
2200 56.298 Docosane 0.5
2500 64.421 Pentacosane 1.3
2600 66.920 Hexacosane 0.2
44
Table 3.3 (Continued)
2700 69.334 Heptacosane 0.8
2900 73.919 Nonacosane 0.2
Total Identified 94.7 a RI = “RetentionIndex” determined in referenceto a homologous series of n-alkanesonan HP-5ms column. bRT=Retention time on column. cThe percentages of each component are reported as raw percentages based on total ion current without standardization. d Identification is tentative and based on mass spectrum only. eAbundant components are indicated in bold. f Correct regioisomer not determined.
Vitispirane cis-Linalool oxide
Palmitic acid (9Z)-Tricosene (Muscalure)
Figure 3.1. Structures of some compounds identified in the Santa Catalina Island, California Opuntia species. All figures reprinted from www.wikipedia.org.
45
Figure 3.1 (Continued)
(E)-Phytol p-Vinylguaiacol
Pristane Phytane
p-Menth-4-en-3-one Geranyl acetone
46
3.2 Compounds Identified in Arizona-Sonora Desert Opuntia Species
Hydrodistillation of cactus specimens from both the Organ Pipe Cactus National
Monument and the Arizona-Sonora Desert Museum produced sufficient extracts for analysis by gas chromatography-mass spectrometry (Table 3.4).
Table 3.4. Essential oil yields of Arizona Opuntia species.
Mass of plant Oil yield and Sample Collection site material description O. acanthocarpa Organ Pipe Cactus 40.0 mg 134.88 g var. major National Monument colorless oil O. acanthocarpa Arizona-Sonora 42.3 mg yellow 58.3 g var. major Desert Museum oil O. phaeacantha var. Organ Pipe Cactus 34.0 mg 149.77 g discata National Monument colorless oil O. phaeacantha var. Arizona-Sonora 110 mg colorless 135.07 discata Desert Museum oil
Fifty-six compounds were identified in O. acanthocarpa var. major growing wild at the Organ Pipe Cactus National Monument (Table 3.5). The primary components of the O. acanthocarpa oil from the Organ Pipe Cactus National Monument included octanoic acid (12.3%), tricosane (10.8%), decanoic acid (10.0%), and 2-hexanol (8.6%).
Alkanes made up 37.7% of the total oil composition, fatty acids and fatty-acid derivatives comprised 28.4%, with only 8.2% terpenoids. Interestingly, the oil also contained 0.5% dimethyl trisulfide. Although dimethyl trisulfide can be harmful if consumed or inhaled by humans, it has been identified in several foods, including garlic (59)(60), cruciferous
47 vegetables (61) (62), and white truffle (63), as well as brewed coffee (64), aged beers
(65), and sake (66).
A total of 48 compounds were identified in the essential oil of O. acanthocarpa var. major growing wild in the Arizona-Sonora Desert Museum (Table 3.6). Chromene compounds composed 71.5% of the total essential oil composition. Eupatoriochromene
(66.6%) dominated the oil composition of the cactus growing in the caliche-rich soil.
Eupatoriochromene is an abundant phytochemical constituent of Eupatorium species
(67), as well as Hemizonia fitchii(68), an annual herb native to California, Centaurea solstitialis, the yellow starthistle (69), and the brittlebush, Encelia farinosa (70).
Additional chromenes also identified in O. acanthocarpa from the Arizona-Sonora
Desert Museum include demethoxyencecelin (3.0%), encecalin (1.6%), and androececalinol (0.3%).
O. phaeacantha var. discata growing wild at the Organ Pipe Cactus National
Monument contained 60 identifiable compounds (Table 3.7). The major components of the oil included tricosane (15.5%), palmitic acid (7.0%), pentacosane (6.3%), cis-linalool oxide (5.1%), trans-linalool oxide (4.8%), and lauric acid (4.5%). Alkane hydrocarbons made up 45.8% of the total oil, with 25.7% fatty acids and derivatives, and only 12.8% terpenoids. O. phaeacantha from the Organ Pipe Cactus National Monument also contained 2.1% homosalate (3,3,5-trimethylcyclohexyl salicylate). Homosalate, a naturally occurring sunscreen that may protect DNA from ultraviolet radiation, has been identified as an emission of both the saguaro cactus (Carnegiea gigantea) and yucca
(Yucca baccata) growing in the Las Vegas, Nevada desert area (71). Homosalate and 2-
48 ethylhexyl salicylate were also detected in O. acanthocarpa from the Arizona-Sonora
Desert Museum (Table 3.5).
Forty-two compounds were identified in the oil of O. phaeacantha var. discata growing wild at the Arizona-Sonora Desert Museum (Table 3.8). The major components identified in the oil include p-vinylguaiacol (16.2%), tributyrin (7.8%), 1-octadecanol
(6.8%), and 1-hexadecanol (6.6%). The essential oil contained 33.2% fatty acids and fatty-acid derivatives and 17.8% alkanes. The major component, p-vinylguaiacol, is a phenolic compound that has been found in several tropical fruits, including in papaya
(72), star apple (73), and mango (74).
The essential oil composition of O. acanthocarpa growing in the drier, lime soil of the Organ Pipe Cactus National Monument differed significantly from that growing in the wetter, caliche soil of the Arizona-Sonora Desert Museum. O. acanthocarpa from the
Arizona-Sonora Desert Museum contained 66.2% eupatoriochromene and consisted of a total chromene concentration of 71.1%, while O. acanthocarpa from the Organ Pipe
Cactus National Monument was devoid of any chromenes. There were 12 n -alkanes common to O. acanthocarpa from both soil types as well as two fatty aldehydes (decanal and dodecanal), and isopropyl palmitate.
Similarly, O. phaeacantha growing in the limey, drier soil of the Organ Pipe
Cactus National Monument differed significantly from that growing in the caliche-rich, wetter soil of the Arizona-Sonora Desert Museum. Notable differences were the relatively abundant concentrations of p-vinylquaiacol, tributyrin, hexadecanol, and phytol in the sample from the Arizona-Sonora Desert Museum site that were either absent or in
49 small concentrations in the Organ Pipe Cactus National Monument site. Additionally, high-molecular-weight alkanes ( C30) were present in the Arizona-Sonora Desert
Museum sample, but absent from the Organ Pipe Cactus National Monument sample.
Conversely, homosalate and isopropyl palmitate were relatively concentrated in the
Organ Pipe Cactus National Monument sample, while the Arizona-Sonora Desert
Museum sample had only a trace amount of isopropyl palmitate and no homosalate.
There were several compounds common to the two samples, including the monoterpenoids, cis- and trans-linalool oxide, linalool, and α-terpineol, the fatty acids decanoic acid, lauric acid, myristic acid, and palmitic acid, and a number of n-alkanes.
The volatile oil yields for both O. acanthocarpa and O. phaeacantha were lower for the samples collected from the Organ Pipe Cactus National Monument (0.030% and
0.023%, respectively) than those collected from the Arizona-Sonora Desert Museum
(0.073% and 0.081%, respectively). Additionally, both O. acanthocarpa and O. phaeacantha oils from Organ Pipe Cactus National Monument had greater concentrations of alkanes than those from the Arizona-Sonora Desert Museum, and fatty acids were more abundant in O. acanthocarpa from Organ Pipe Cactus National Monument. For O. phaeacantha, fatty acid and terpenoid concentrations were similar for the two collection sites.
50
Table 3.5: Chemical composition of the essential oil of Opuntia acanthocarpa var. major from Organ Pipe Cactus National Monument.
RIa RTb Compound %c
801 3.613 Hexanal trd
810 3.756 2-Hexanol 8.6
836 4.299 Furfural 1.7
838 4.357 3-Methyl-2-hexanone trd
871 5.144 n-Hexanol 1.0
893 5.867 2-Heptanone 1.6
900 6.146 2-Heptanol trd
903 6.201 Heptanal 0.9
908 6.483 Butyl propanoate 0.2
918 7.037 Prenyl acetate 0.3
965 8.502 Benzaldehyde 0.5
973 8.855 Dimethyl trisulfide 0.5
994 9.819 2-Pentylfuran 0.5
1034 11.755 Unidentifiede 1.0
1044 12.185 Benzeneacetaldehyde 0.9
1073 13.548 cis-Linalool oxide (furanoid) 1.5
1088 14.301 trans-Linalool oxide (furanoid) 0.8
1106 15.084 Decanal 1.0
1127 16.071 α-Campholenal 0.7
51
Table 3.5 (Continued)
1166 17.950 Camphenone 1.0
1179 18.602 Octanoic acid 12.3
1274 23.145 Vitispirane 0.5
1280 23.276 Nonanoic acid 0.6
1300 24.212 Tridecane 0.8
1377 28.097 Decanoic acid 10.0
1400 28.569 Tetradecane 1.0
1411 28.951 Dodecanal 0.3
1464 31.142 2,6,10-Trimethyltridecane 1.4
1500 32.969 Pentadecane 2.0
1552 34.621 Nonylcyclohexane 0.7
1566 35.178 2-Methylpentadecane 0.4
1578 35.635 Dodecanoic acid 1.7
1600 36.597 Hexadecane 2.1
1615 37.068 Tetradecanal 0.5
1652 38.418 2,6,10-Trimethylpentadecane (= Norpristane) 0.5
1700 40.293 Heptadecane 0.7
1708 40.503 2,6,10,14-Tetramethylpentadecane (= Pristane) 0.6
1812 44.132 2,6,10,14-Tetramethylhexadecane (= Phytane) 0.6
1884 46.621 1-Nonadecene 0.7
2000 50.305 Eicosane 0.3
2030 51.134 Isopropyl palmitate 1.1
52
Table 3.5 (Continued)
2089 53.175 1-Heneicosene 0.9
2100 53.460 Heneicosane 3.5
2199 56.125 1-Docosene 0.9
2200 56.388 Docosane 2.0
2300 59.013 Tricosane 10.8
2398 61.772 1-Tetracosene 2.1
2400 61.912 Tetracosane 4.1
2459 63.484 Unidentifiedf 1.3
2464 63.614 Unidentifiedg 0.9
2495 64.362 1-Pentacosene 0.8
2500 64.523 Pentacosane 4.9
2599 66.887 1-Hexacosene 0.9
2600 67.005 Hexacosane 0.7
2700 69.435 Heptacosane 3.5
2800 71.650 1-Octacosene 0.2
2800 71.747 Octacosane 0.3
53
Table 3.5 (Continued)
2833 72.393 Squalene 0.3
2900 74.007 Nonacosane 0.7
Total Identified (56) 96.9 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%). e MS, m/z(%): 139(9), 137(14), 119(21), 117(64), 112(70), 110(100), 77(27), 75(38), 71(61), 65(13), 63(13), 55(16), 43(93). f MS, m/z(%): 280(14), 279(54), 163(18), 137(14), 123(21), 111(20), 109(29), 105(23), 97(27), 95(50), 85(25), 83(27), 81(54), 71(27), 69(100), 67(57), 57(39), 55(48), 43(30), 41(45). g MS, m/z(%): 280(7), 263(15), 245(7), 179(8), 165(10), 163(11), 151(11), 137(11), 125(18), 111(30), 109(18), 105(18), 97(46), 95(23), 85(26), 83(46), 81(22), 71(33), 69(100), 67(25), 57(50), 55(46), 43(36), 41(39).
Table 3.6. Chemical composition of the essential oil of Opuntia acanthocarpa var. major from Arizona-Sonora Desert Museum.
RIa RTb Compound %c
1190 19.292 α-Terpineol trd
1206 19.877 Decanal trd
1300 24.155 Tridecane 0.1
1375 27.426 α-Copaene trd
1392 28.143 -Elemene 0.2
1410 28.904 Dodecanal 0.1
1418 29.259 (E)-Caryophyllene 0.1
1438 30.071 Aromadendrene 0.3
54
Table 3.6 (Continued)
1478 31.655 γ-Muuolene 0.2
1484 31.930 ar-Curcumene 0.1
1495 32.393 Valencene 0.2
1500 32.621 Pentadecane 0.5
1510 32.973 -Bisabolene 0.3
1514 33.164 γ-Cadinene 0.1
1524 33.536 δ-Cadinene 0.2
1550 34.563 Elemol 0.2
1566 35.148 (E)-Nerolidol 1.2
1570 35.398 (3Z)-Hexenyl benzoate 0.1
1578 35.644 Spathulenol 2.5
1583 35.851 Caryophyllene oxide 1.1
1600 36.517 Hexadecane 0.3
1619 37.199 Unidentifiede 1.8
1631 37.948 γ-Eudesmol 0.2
1651 38.377 Demethoxyencecalin 3.0
1655 38.548 Selin-11-en-4α-ol 1.1
1678 39.428 Androencecalinol 0.3
1700 40.219 Heptadecane 1.4
1715 40.741 Pentadecanal 0.2
1766 42.606 Eupatoriochromene 66.2
1800 43.747 Octadecane 0.7
55
Table 3.6 (Continued)
1803 43.930 2-Ethylhexyl salicylate 0.6
1812 44.216 Unidentifiedf 0.8
1839 45.275 6,10,14-Trimethylpentadecan-2-one 0.4
1870 46.065 Encecalin 1.6
1885 46.563 Homosalate 0.7
1888 46.666 Unidentifiedg 1.1
1900 47.084 Nonadecane 0.2
1923 48.050 Unidentifiedh 2.4
1937 48.509 Unidentifiedi 1.7
2000 50.285 Eicosane 0.1
2029 51.108 Isopropyl hexadecanoate 0.7
2076 52.527 Octadecanol 0.1
2100 53.362 Heneicosane 0.8
2117 53.981 (E)-Phytol 1.1
2200 56.282 Docosane 0.2
2277 58.335 (9Z)-Tricosene (= Muscalure) 0.2
2300 59.100 Tricosane 1.2
2400 61.807 Tetracosane 0.4
2457 63.417 2-Methyltetracosane 0.2
2500 64.403 Pentacosane 0.4
2600 66.908 Hexacosane 0.1
2700 69.330 Heptacosane 1.2
56
Table 3.6 (Continued)
2900 73.920 Nonacosane 0.5
Total Identified (48) 91.5 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%). e MS, m/z(%): 206(19), 162(100), 147(59), 134(20), 133(21), 121(24), 120(41), 119(27), 108(31), 107(50), 106(56), 105(52), 95(30), 93(58), 91(68), 81(56), 79(56), 77(33), 67(41), 55(34), 53(21), 43(13). f MS, m/z(%): 234(14), 220(14), 219(100), 203(20), 201(40), 187(21), 159(9), 115(9), 91(12), 79(9), 77(9), 69(11), 55(12), 43(25), 41(13). g MS, m/z(%): 216(81), 201(100), 173(21), 138(6), 121(7), 115(21), 109(13), 97(17), 95(13), 91(16), 83(23), 69(38), 57(21), 55(31), 43(44). h MS, m/z(%): 218(54), 203(100), 185(5), 147(8), 133(6), 105(6), 77(7), 69(10), 55(10), 43(31), 41(8). i MS, m/z(%): 219(3), 187(5), 159(4), 147(5), 135(16), 134(29), 121(12), 119(18), 107(38), 93(53), 91(12), 81(21), 79(20), 71(36), 67(31), 55(26), 43(100), 41(18).
Table 3.7. Chemical composition of the essential oil of Opuntia phaeacantha var. discata from Organ Pipe Cactus National Monument.
RIa RTb Compound %c
758 3.551 4-Methyl-2-pentanol trd
836 4.296 Furfural trd
1044 12.191 Phenylacetaldehyde trd
1067 13.232 Acetophenone 1.3
1072 13.578 cis-Linalool oxide (furanoid) 5.1
1088 14.327 trans-Linalool oxide (furanoid) 4.8
1102 14.879 Linalool 0.6
1186 18.935 p-Cymen-8-ol 1.0
57
Table 3.7 (Continued)
1190 18.996 Octanoic acid tr
1191 19.163 α-Terpineol 0.7
1307 24.847 p-Vinylguaiacol 0.5
1309 24.942 (2E,4E)-Decadienal 0.4
1345 26.721 Benzalacetone 0.8
1368 27.867 Decanoic acid 3.0
1429 30.956 (E)-β-Famesene 0.1
1431 31.052 6-Amyl-α-pyrone 0.4
1446 31.818 Massoilactone 0.6
1488 32.155 Maltyl isobutyrate 3.1
1499 32.532 α-Zingiberene 1.9
1500 32.685 Pentadecane 0.7
1509 32.991 (E,E)-α-Farnesene 1.1
1569 35.377 Lauric acid 4.5
1600 36.738 Hexadecane 0.7
1666 38.940 2-Methylhexadecane trd
1700 40.296 Heptadecane 0.9
1772 42.753 Myristic acid 1.3
1800 43.814 Octadecane 0.5
1836 44.952 Butyl salicylate butyl ether 0.4
1861 45.641 Tributyrin 0.6
1888 46.653 Homosolate 2.1
58
Table 3.7 (Continued)
1900 47.162 Nonadecane 1.0
1932 48.041 Methyl palmitate 0.3
1953 48.679 1-Heptadecanol 0.5
1972 49.605 Palmitic acid 7.0
2000 50.379 Eicosane 0.3
2031 51.154 Isopropyl palmitate 3.5
2068 52.309 2-Methyleicosane 0.6
2101 53.273 Methyl linoleate 0.4
2100 53.469 Heneicosane 3.6
2142 54.716 Linoleic acid 1.1
2148 54.878 Oleic acid 1.4
2199 56.187 1-Docosene 0.5
2200 56.363 Docosane 2.4
2227 57.087 (2Z)-Docosene 0.4
2280 58.304 (9Z)-Tricosene (= Muscalure) 1.1
2300 58.987 Tricosane 15.5
2397 61.771 1-Tetracosene 1.9
2400 61.928 Tetracosane 4.0
2494 64.350 1-Pentacosene 0.6
2500 64.548 Pentacosane 6.3
2531 65.269 13-Methylpentacosane 0.8
2598 66.871 1-Hexacosene 0.8
59
Table 3.7 (Continued)
2600 66.985 Hexacosane 0.9
2636 67.799 13-Methylhexacosane 0.8
2700 69.413 Heptacosane 4.1
2739 70.227 Methyl lignocerate 1.1
2800 71.754 Octacosane 0.3
2841 72.588 Hexacosanal 1.0
2900 73.997 Nonacosane 0.8
2910 74.120 3,7-Dimethyloctacosane 1.4
Total Identified (60) 100.0 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Rentention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. dtr = “trace” (< 0.05%).
Table 3.8. Chemical composition of the essential oil of Opuntia phaeacantha var. discata from Arizona-Sonora Desert Museum.
RIa RTb Compound %c
1072 13.676 cis-Linalool oxide (furanoid) 3.4
1091 14.427 trans-Linalool oxide (furanoid) 3.5
1104 15.004 Linalool 0.9
1194 19.292 α-Terpineol 1.3
1196 19.416 Octanoic acid trc
60
Table 3.8 (Continued)
1256 22.322 Geraniol 0.6
1287 23.611 Nonanoic acid 0.9
1310 24.659 Undecanal 0.4
1316 25.029 p-Vinylguaiacol 16.2
1380 27.627 Decanoic acid 5.7
1413 29.055 Dodecanal 0.2
1500 32.785 Pentadecane 0.3
1516 33.223 Tridecanal 0.5
1584 35.885 Lauric acid 5.9
1677 39.406 Tridecanoic acid 0.5
1773 42.921 Myristic acid 1.7
1861 45.779 Tributyrin 7.8
1887 46.666 1-Hexadecanol 6.6
1900 47.261 Nonadecane 0.8
1934 48.415 (2Z)-Nonadecene 0.3
1971 49.572 Palmitic acid 3.0
2035 51.282 Isopropyl palmitate 1.1
2072 52.407 2-Methyleicosane 0.3
2087 53.099 1-Octadecanol 6.8
2100 53.541 Heneicosane 2.0
2117 53.982 (E)-Phytol 5.2
2274 58.247 2-Methyldocosane 0.5
61
Table 3.8 (Continued)
2284 58.526 (9Z)-Tricosene (= Muscalure) 3.1
2296 59.045 1-Tricosene 1.0
2300 59.293 Tricosane 2.8
2500 64.586 Pentacosane 1.7
2584 66.506 Hexadecyl octanoate 0.9
2595 66.778 Unidentifiede 3.9
2600 67.079 Hexacosane 0.6
2700 69.499 Heptacosane 1.1
2800 71.836 Octacosane trd
2900 74.093 Nonacosane 0.8
2994 75.972 1-Triacontene 0.6
3000 76.270 Triacontane 0.8
3100 78.391 Untriacontane 1.6
3200 80.666 Dotriacontane 1.6
3351 83.379 5-Methyltriacontane 1.4
3600 88.049 Hexatriacontane 1.7
Total Identified (42) 96.1 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%). e MS, m/z(%): 299(20), 298(100), 283(3), 251(3), 233(4), 223(5), 195(3), 165(5), 151(4), 137(3), 115(3), 89(3), 77(3), 51(1).
62
Octanoic acid Eupatoriochromene
Encecalin Tributyrin
Homosalate α-Terpineol
Figure 3.2. Structures of some compounds identified in Arizona Sonora Desert Opuntia species. Figures reprinted from www.webbook.nist.gov.
63
3.3 Essential oil composition of green Stenocereus thurberi
The volatile composition of green S. thurberi was analyzed (Table 3.9). S. thurberi was composed of 32.8% alkane, 27.5% alkene, 13.1% ester, and 8.0% aldehyde.
The compound in green S. thurberi oil with the greatest percentage of the total composition was 1-eicosene (6.7%). The other major components of the green specimen were: isopropyl hexadecanoate (6.6%), tetracosane (5.4%), decanoic acid (5.0%), 1- nonadecene (4.6%), and 1-heneicosene (4.1%). The green volatiles contained 2.9% lapachol and 0.8% 1-epi-cubenol. Lapachol, a naphthoquinone, has previously only been found in the wood of certain tropical plants such as Diphysa rubinoides, a tree native to
Vera Cruz, Mexico (75). Although lapachol has only been identified in South American trees, S. thurberi has a wooden skeleton within each arm which may be the site of lapachol production. Lapachol has been found to be active against certain cancerous tumors (76)(77).
Table 3.9. Chemical composition of green Stenocereus thurberi Engelm. obtained from the Organ Pipe Cactus National Monument.
RIa RTb Compound % S.
thurberic
1106 15.121 Nonanal 1.4
1206 19.896 Decanal 1.1
1285 23.535 Bornyl Acetate 0.9
64
Table 3.9 (Continued)
1323 25.328 Methyl decanoate 0.9
1366 27.284 Decanoic Acid 5.00
1511 33.077 Tridecanal trd
1526 33.665 Methyl Dodecanoate 1.1
1566 35.191 Dodecanoic Acid 2.1
1588 36.064 1-Hexadecene trd
1601 36.565 Hexadecane trd
1614 37.029 Tetradecanal 2.3
1632 37.770 1-epi-Cubenol 0.8
1685 39.761 2-Methyl-1-hexadecene 1.6
1693 39.988 1-Heptadecene 1.9
1700 40.239 Heptadecane 0.9
1721 41.025 (2Z)-Heptadecene trd
1744 41.828 Unidentified 1.1
1763 42.503 Unidentified 0.7
1791 43.534 1-Octadecene 3.7
1798 43.778 Octadecane 2.1
1813 44.273 Hexadecanal 1.2
1826 44.687 Isopropyl myristate 0.9
1839 45.310 Methylhexadecanal 2.0
1880 46.416 3-Heptadecanone 0.7
1896 46.905 1-Nonadecene 4.6
65
Table 3.9 (Continued)
1903 47.126 Nonadecane 3.6
1921 48.000 Methyl hexadecanoate 1.9
1929 48.273 (2Z)-Nonadecene 0.9
1997 50.136 1-Eicosene 6.7
2003 50.334 Eicosane 2.0
2013 50.655 Hexadecyl acetate 0.8
2029 51.146 Isopropyl hexadecanoate 6.6
2090 53.210 1-Heneicosene 4.1
2096 53.397 Heneicosane 2.8
2116 53.967 Lapachol 2.9
2167 55.219 Unidentified 1.1
2173 55.405 Elaidic acid 1.2
2196 56.069 1-Docosene 0.4
2200 56.325 Docosane 2.1
2294 58.989 1-Tricosene trd
2300 59.143 Tricosane 2.2
2395 61.711 1-Tetracosene 2.3
2400 61.840 Tetracosane 5.4
2493 64.325 1-Pentacosene 1.2
2500 64.447 Pentacosane 2.6
2600 66.963 Hexacosane 1.3
2700 69.373 Heptacosane 1.7
66
Table 3.9 (Continued)
2800 71.702 Octacosane 0.5
2900 73.691 Nonacosane 3.2
3000 76.100 Tricontane 1.0
3100 78.258 Untriacontane 1.3
% Identified 97.1
a RI = “Retention Index” determined in reference to a hoRImologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%).
3.4 Compounds Identified in Necrotic and Lab Rot Opuntia littoralis and Stenocereus thurberi
All of the necrotic samples contained much larger percentages of volatile compounds than the green specimens. The green cactus was non-toxic to D. melanogaster and, in fact, was used to scale-up the number of flies available for testing.
The green cactus had very low concentrations of volatile components. Both the necrotic
O. littoralis and the necrotic S. thurberi were lethal to the fruit flies. The necrotic specimens yielded a much larger concentration of volatiles. It is possible that the green cactus is non-toxic to D. melanogaster because the flies are able to obtain nutrition from the cactus while ingesting small concentrations of toxic volatiles. The necrotic specimens may be lethal to fruit flies because the flies obtain a high concentration of toxic volatiles.
67
It is suggested that the natural rot of O. littoralis is a quick process (78).
Approximately 1.5 kg of O. littoralis was received from the Catalina Island Conservancy in Southern California. Of the specimens, different stages of necrosis were observed.
The green cactus pad has a clear, thick epidermis with a green biomass. The first step in necrosis is the drying and darkening of the epidermis of the pad. The entire pad obtains a bark-like appearance (Figure 3.1). As the epidermis decays to “bark”, the green biomass turns to yellow and white (Figure 3.2). The yellow and white color turns caramel and dark brown at the late-rot stage (Figure 3.3). The natural rot is thick and dry. An individual pad contains multiple states of necrosis. The necrosis starts at the joint where the pad is attached to the cactus and spreads inward. A single pad contains necrotic tissue with all stages of rot from the yellow/white seen at the beginning of the rot to the dark brown late rot tissue.
Figure 3.3. Early field rot O. Figure 3.4. Bark-like exterior Figure 3.5. Late natural rot littoralis. of natural rot of O. littoralis. of O. littoralis.
68
The field rot dynamic headspace of O. littoralis specimens did not contain any oil other than 1-docosene. Additionally, the O. littoralis field rot analysis revealed mere traces of carboxylic acids. These results are surprising because almost 1.5 kg of sample were tested. Multiple extracts produced no essential oil from the specimen. Recently, a study found that lab-rot O. littoralis contained 18 compounds in its headspace, which was measured with solid phase micro extraction (SPME) (78) . The study also investigated the attraction of D. mojavensis to progressive phases of cactus necrosis (78). But, this investigation used just 70g of cactus of a lab-rotted specimen that utilized 7 different species of microbes to produce the rot. The number of microbes colonizing a natural rot could greatly exceed the 7 different microbe species used in the lab rot study.
Additionally, lab rot specimens are replete with water and have a slushy consistency.
The natural rot was dry without any exudate. It is possible that the essential oil is released during the necrotic process. Interestingly, the previous study found cactus flies to be equally attracted to 1-week and 5-week rots and less attracted to 9-week rot. It may be that the oil content is higher in early rots as compared to late rots, and, the higher the oil concentration, the more flies that are attracted to the rot.
The major compounds of lab rot O. littoralis are: hexanoic acid (75.9%), octanoic acid (8.8%), decanoic acid (3.4%), and pentanoic acid (1.7%) (Table 3.10). Hexanoic acid and octanoic acid have been shown to be toxic to Drosophila melanogaster (79)
(80). Hexanoic acid has been found to cause reversible coma in D. melanogaster and octanoic acid to kill D. melanogaster (79). Decanoic acid has been shown to be non- toxic to D. melanogaster (79). Phenethyl alcohol was found to be present in the lab rot at concentration of 0.2 % in O. littoralis. Phenethyl alcohol is readily oxidized to
69
phenylacetaldehyde by NAD+-dependent alcohol and aldehyde dehydrogenases (81).
Phenethyl alcohol was found to not cause genotoxic effects in D. melanogaster (82)
Table 3.10. Essential oil composition of lab-rot O. littoralis.
RIa RT Compound % Lab-rot
Columnb O. litteralis c
763 3.791 Butanoic acid 1.0
861 4.915 3-Methyl butanoic acid 0.1
880 5.440 Pentanoic acid 1.7
967 10.700 Hexanoic acid 75.9
1074 16.466 Heptanoic acid 0.1
1136 16.507 2-Phenylethyl alcohol 0.2
1167 17.253 Octanoic acid 8.8
1255 22.116 Isopentyl hexanoate 0.1
1261 22.399 2-Phenylethyl acetate 0.2
1267 22.700 3,5-Dimethylbenzenemethanol 0.1
1270 22.925 Nonanoic acid 0.1
1289 23.703 Isobornyl acetate trd
1292 23.845 (2E,4Z)-Decadienal 0.1
1301 24.248 Cyclopentyl hexanoate 0.1
1304 24.516 2-Furancarboxylic acid, tetrahydro-3-methyl-5oxo 0.2
1316 25.036 (2E,4E)-Decadienal trd
70
Table 3.10 (Continued)
1326 25.264 Methyl decanoate 0.1
1354 25.422 2-Phenylethyl propionate 0.1
1359 26.550 Eugenol trd
1364 26.781 Nonalactone trd
1369 26.993 Dihydro eugenol trd
1369 27.196 Decanoic acid 3.4
1401 27.313 3-Decenoic acid trd
1440 28.536 4-Hexenoic acid, 6-(acetyloxy)-4-methyl trd
1442 30.141 2-Phenethyl butanoate 0.3
1450 30.230 Pentyl octanoate trd
1455 30.545 (Z)-6,10-Dimethyl-5,9-undecadien-2-one trd
1464 30.789 Undecanoic acid 0.1
1470 31.152 γ-Decalactone 0.1
1483 31.391 2,6-Dimethylnon-1-en-3yn-5yl-butanoate trd
1494 32.076 Phenylethyl isovalerate trd
1499 32.357 2-Tridecanone trd
1510 32.537 Tridecanal 0.1
1522 33.233 (2E,4E) Dodecadienol trd
1528 33.452 Methyl dodecanoate 0.1
1530 2(4H(-Benzofuranone, 56,6,7,7a-tetrahydro-4,4,7a trd 33.690 trimethyl-
1544 33.769 2-Phenylethyl pentanoate 0.1
71
1554 34.307 3,7,11-Trimethyl-1-dodecanol 0.1
1566 34.711 Dodecanoic acid 2.0
1598 35.185 Ethyl dodecanoate trd
1606 36.412 2,6-Dimethoxy-4-(2-propenyl)-phenol trd
1615 36.704 Tetradecanal 0.1
1627 37.043 6-ethyl-3-octyl Hexanoate trd
1635 37.525 1-epi-Cubenol trd
1644 37.869 2-Phenylethyl hexanoate 0.2
1646 38.155 Isopentyl pentadecanoate trd
1657 38.277 2-Acetoxy-1,1,10-trimethyl-6,9-epioxydecalin trd
1662 38.641 (2E)-Tridecenoic acid trd
1665 38.876 Tridecanoic acid trd
1680 38.986 γ-Dodecalactone 0.1
1686 39.494 1-Heptadecene 0.2
1700 39.712 Heptadecane 0.1
1727 41.193 Methyl tetradecanoate trd
1738 41.450 (E)-Sesquilavandulyl acetate trd
1744 41.833 2-Phenylethyl heptanoate trd
1765 42.582 Tetradecanoic acid 0.2
1787 43.407 Methyl Pentadecanoate trd
1796 43.674 Methyl 12-methyl-tetradecanoate 0.1
1799 43.787 1-Tetradecyl acetate trd
1804 43.942 (7Z)-Hexadecenal trd
72
Table 3.10 (Continued)
1809 44.127 9-Methyl-Z-10-pentadecen-1-ol trd
1814 44.279 Hexadecanal 0.1
1827 44.700 Isopropyl tetradecanoate 0.1
1846 45.304 2-Heptadecanone 0.1
1869 45.572 Pentadecanoic acid 0.1
1892 45.915 n-Hexadecanol 0.2
1900 46.783 2-Methyl hexadecanol 0.1
1910 47.027 1,2-Epoxyoctadecane trd
1915 47.635 Nonyl phenyl carbonate 0.1
1921 47.795 Methyl hexadecanoate 0.1
1930 48.003 16-Hexadecanolide trd
1943 48.276 Phytol trd
1963 48.686 Hexadecanoic acid 0.8
1991 49.343 Ethyl hexadecanoate 0.1
2000 50.194 Eicosane trd
2030 50.771 Isopropyl hexadecanoate trd
2091 51.148 Methyl linoleate 0.1
2097 53.216 Methyl oleate 0.1
2102 53.407 (11Z)-Octadecenoic acid 0.1
2117 53.537 Lapachol 0.1
2167 54.153 Ethyl linoleate 0.2
2174 55.226 Ethyl oleate 0.1
73
Table 3.10 (Continued)
2183 55.413 Tetradecyl hexadecanoate trd
2198 55.671 1-Docosene trd
2200 56.092 Docosane trd
2209 56.325 Methyl (10Z)-Nonadecenoate trd
2281 56.572 Pentadecyl hexanoate 0.1
2296 58.613 Nonadecyl acetate trd
2300 59.021 Tricosane trd
2308 59.141 (2E)-Tricosene trd
2365 59.368 Eicosanoic acid 0.1
2383 60.907 Hexadecyl hexanoate trd
2395 61.373 1-Tetracosene trd
2400 61.704 Tetracosane trd
2492 61.838 1-Pentacosene trd
2500 64.312 Pentacosane trd
2596 64.443 1-Hexacosene trd
2600 66.832 Hexacosane trd
2700 66.951 Heptacosane trd
2800 69.365 Octacosane trd
2831 72.356 Squalene trd
2900 73.949 Nonacosane trd
74
Table 3.10 (Continued)
3000 76.109 Triacontane 0.1
3100 78.255 Untriacontane 0.1
% Identified 99.7
a RI = “Retention Index” determined in reference to a hoRImologous series of n-alkanes on an HP-5ms column. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%).
The necrosis of S. thurberi may be a slow, gradual process. Although the cactus
specimens were damaged during a torrential rain that blew them down during September
2012, the specimens were not received for analysis until March 2013. Despite the
passing of more than 5 months, 2 of the 7 arms of cacti were not fully necrotic. Receipt
of varying stages of necrosis allowed the necrosis process to be observed and
documented. Green S. thurberi has a thick yellow biomass within a tough, translucent
epidermis. The color of the biomass changes from yellow to caramel (Figure 3.4). The
caramel then turns dark brown (Figure 3.5). During this change, the epidermis obtains a
white color and separates from the biomass.
75
Figure 3.6. Early natural rot S. thurberi. Figure 3.7. Late natural rot S. thurberi,
The two early necrotic field rots obtained from the Organ Pipe Cactus National
Monument were analyzed by GC-MS (Table 3.11). The early rot composition is very close to the lab rot oil extract, which is dominated by carboxylic acids. 84.8% of the total oil components were carboxylic acids. Butanoic acid dominated the oil consisting of
33.0% of the total oil fraction. Hexanoic acid (16.2%), octanoic acid (15.6%), and nonanoic acid (11.1%) comprise 42.9% of the total oil fraction. ο-Guaiacol comprised
2.2% of the early rot. ο-Guaiacol is a precursor to eugenol (83), which comprises 0.4% of the late rot of S. thurberi.
76
Table 3.11. Volatile composition of early natural rot S. thurberi Engelm.
RI RT Compound % Early rot S. thurberi
809 3.761 Butanoic acid 33.1a
836 4.309 Furfural 1.7
855 8.376 Isopropyl butanoate 0.1
966 8.574 Benzaldehyde 0.1
991 9.711 Phenol 0.4
1000 10.118 Butyl butanoate 1.1
1006 10.415 Hexanoic acid 16.3a
1046 12.277 Benzeneacetaldhyde 2.2
1059 12.897 Pentyl butanoate 5.9
1092 14.466 ο-Guaiacol 2.2
1097 14.675 (2Z)-Pentenyl Butanoate 0.2
1120 15.770 Phenylethyl alcohol 0.2
1180 18.647 Terpinen-4-ol 0.1
1195 19.371 Methyl salicylate 1.6
1264 22.563 Octanoic acid 15.6a
1265 22.612 Nonanoic acid 11.1a
1348 26.306 Benzyl butanoate 0.3
1386 27.951 Decanoic acid 6.1
1445 30.338 2-Phenylethyl butanoate 0.3
1502 32.704 Pentadecane 0.1
77
Table 3.11 (Continued)
1584 35.893 Dodecanoic acid 0.9
1602 36.599 Hexadecane 0.2
2369 61.005 1-Heneicosanol 0.2
% Identified 100%
aAbundant components are indicated in bold.
The primary constituents of dynamic headspace collection of volatiles from the naturally necrotic late specimen of S. thurberi are : p-cresol (32.6%), heptacosane
(7.1%), and hexacosane (7.0%)(Table 3.12). The volatiles collected from the dynamic headspace of the naturally necrotic specimen is composed of 48% alkane and just 2.2% carboxylic acid. The natural rot is dominated by p-cresol (55.9%). The natural-rot specimen contained just 3.8% carboxylic acid and the dynamic headspace contained
2.2%, which is the concentration of hexadecanoic acid identified. The natural rot of S. thurberi was void of any alkanes, which dominated the dynamic headspace of the rot.
p-Cresol, the dominant component of both the dynamic headspace and the late, natural rot extracts, is mainly used in the formulation of antioxidants for lubricating oil and motor fuels, rubber, polymers, elastomers and food products (84). Although p- cresol has been found to not be linked to sex-linked recessive lethal mutations in D. melanogaster (85), dermal or oral exposure to rats can produce severe toxicity and may result in death (84). A 600 mg/kg dose of p-cresol causes death in rats (84). Anisole has been identified as an odorant evoking a strong response in D. melanogaster olfactory
78 circuitry (86). Benzaldehyde was identified in the field rot (0.5%) of S. thurberi.
Genotoxicity testing of benzaldehyde using the Wing Spot Assay at concentrations of
0.2M showed significant microscopic changes in the phenotype in terms of orange discoloration of the thorax and darkening of the eye color (87). Benzaldehye is the most toxic of the identified compounds tested in this study. The natural rot contained 2- heptanol (0.8% dynamic headspace) and 2-nonanol (1.1% dynamic headspace/1.7% rot).
Heptanol and nonanol produce dose-independent responses, with larvae being attracted to heptanol and repulsed by nonanol (88). Eugenol is present in the rot extract at just 0.4%.
Eugenol has been found to be toxic to D. melanogaster with a LD50=1.90µg/fly (89).
Phenol was identified in the natural rot dynamic headspace of S. thurberi (0.3%).
Phenol has been shown to extend the process of metamorphosis and decrease the total number of offspring in D. melanogaster (90). Long-term feeding of phenol to fruit flies causes an increase in the resistance to phenol toxicity, lowering the benzene toxicity, and inducing enzymatic activities of glutathione S-transferases (91) Phenol does not increase the frequency of sex-linked recessive lethal mutations in D. melanogaster (92).
The volatiles obtained by dynamic headspace collection from the late, natural rot consisted of 19 identifiable alkanes containing 12 carbons or more, which accounted for
48.5 % of the dynamic headspace fraction. The following compounds identified in the natural rot have been found to be sexual pheromones in D. melanogaster: tricosane
(1.8%), pentacosane (6.4%), methyl-hexacosane (2.4%), heptacosane (7.1%), and methyl-octacosane (0.7%) (93). These long alkane compounds may attract the D. mojavensis flies to the cactus rot.
79
The late, natural rot of S. thurberi contained 2.2% palmitic acid in the dynamic airspace and 2.5% in the rot. Oleic acid constituted 0.2% of the natural rot. Palmitic acid and oleic acid are saturated fatty acids found in D. melanogaster during normal development (94). Furfural (0.2% of rot), an aldehyde, stimulates an increase in sex- linked recessive lethal mutations in germ cells of D. melanogaster (95).
The late, natural rot consisted of 4.5% ketone in the dynamic headspace and 8.7% in the rot itself. 2-Nonanone (1.1% dynamic headspace/1.2% rot) protects D. melanogaster by antagonist binding with an odorant receptor preventing methanethiosulonate action at the receptor (96). Methanethiosulfonate is a small compound that blocks cysteines and other sulfhydryl groups enabling the study of enzyme activation and protein function. Isophorone (0.5% of rot) is used as a solvent for resins, polymers and pesticide formulations (97). Although isophone does not induce sex-linked recessive lethal mutations in D. melanogaster (97), it does interfere with glutathione (GSH) levels in rat liver and male reproductive organs (98). 2-Undecanone, which is listed as a pesticide with the U.S. Environmental Protection Agency, induces glutathione S-transferase (GST1) [GST1 is a detoxifying enzyme] in Drosophila (99). 2-
Tridecanone, a methylketone insecticide, constituted 0.6% of the natural rot of S. thurberi.
.
80
Table 3.12. Esential oil composition of late, field-rot Stenocereus thurberi Engelm.
RIa RTb Compound % % Stenocereus Stenocereu thurberi s thurberi dynamic natural headspacec rotc
808 3.729 Hexanal 0.4
846 4.555 Furfural 0.2
867 5.067 (3Z)-Hexenol 0.8
878 5.397 Hexanol 0.2
899 5.882 2-Heptanone 3.1 0.4
902 6.182 2-Heptanol 0.8
907 6.474 Heptanal 0.9
909 6.540 Unidentified 0.4
965 8.537 6-Methyl 2-Heptanone 0.6
971 8.781 Benzaldehyde 0.5
986 9.501 Unidentified 0.2
987 9.555 Phenol 0.3
1000 10.123 Unidentified 1.2
1026 11.115 p-methyl-Anisole 0.7 4.5
1030 11.548 2-Ethyl hexanol 0.3
1089 14.108 p-Cresol 32.6 55.9
1119 14.401 Diethyl carbitol 0.8 0.4
1123 14.643 2-Nonanone 1.1 1.2
81
Table 3.12 (Continued)
1125 16.037 1-Nonen-4-ol 0.4
1130 16.261 2-Nonanol 0.4 1.7
1133 16.389 (E) 3-5-Heptadien-2-one, 6-methyl 0.2
1135 16.492 3,4-Dimethylcyclohexanol 0.1
1139 16.691 2-Phenylethyl alcohol 0.3
1146 17.010 Isophorone 0.5
1164 17.784 keto-Isophorone 0.2
1166 17.962 Veratrol 0.4
1175 18.396 2-Decanone 0.4
1188 19.061 1-Nonanol 0.2
1192 19.215 Ethanol, 2-(2-butoxyethoxy)- 1.2
1195 19.379 2-Isobutyl-3-methoxypyrazine 0.2
1202 19.738 Unidentified 0.6
1207 19.977 Creosol 1.3
1226 21.055 Unidentified 0.4
1252 22.017 2,3-Dimethoxytoluene 0.3
1256 22.190 Unidentified 0.2
22.339 2-Cyclohexen-1-one, 5-methyl-2- 1259 (1-methylethyl)- 0.1
1281 23.332 Isobutyl benzoate 0.4
1291 23.791 p-Ethylguaiacol 0.7
1295 23.972 2-Undecanone 0.3 0.9
82
Table 3.12 (Continued)
1314 24.875 2-Undecanol 1.8
1326 25.445 Unidentified 0.8
1336 25.788 Benzene, 4-ethyl-1,2-dimethoxy- 0.6
1362 26.905 Phenylethyl alcohol, propionate 0.1
1370 27.243 Eugenol 0.4
1385 27.865 α-Copaene 0.2
1418 29.254 Methyleugenol 0.4
1431 29.797 Unidentified 0.7
1468 31.317 Ionol <α-isomethyl-(E)-> 4.9
1481 31.864 Unidentified 0.6
32.173 3-Buten-2-ol, 2-methyl-4-(1,3,3- trimethyl-7-oxabicyclo 1489 [4.1.0]hept-2-yl)- 1.3
1509 32.946 2-Tridecanone 0.6
1517 33.271 2-Tridecanol 0.5
1526 33.626 Unidentified 0.9
1533 33.900 Unidentified 0.1
1555 34.786 Lippifoli-1(6)-en-5-one 1.4
1568 35.375 Elemicine 0.2
1611 36.927 Longiborneol 1.2
1690 39.883 1-Heptadecene 0.7
1716 40.778 Curcuphenol 0.3
1730 41.210 Hetpyl phenyl carbonoate 0.3
83
Table 3.12 (Continued)
1735 41.497 (2Z,6E )Farnesol 0.2
1869 46.056 Benzoic acid, 2-phenylethyl ester 0.4
1886 46.599 Hexadecanol 2.2
1896 46.910 1-Nonadecene 0.7
1966 49.447 Palmitic acid 2.2 2.5
2085 53.049 n-Octadecanol 2.0
2099 53.471 Unidentified 0.5
2170 55.309 Elaidic acid 0.8
2173 55.404 Oleic acid 0.2
2177 55.936 Octadecanoic acid 0.3
2200 56.375 Docosane 1.0
2300 59.222 Tricosane 1.8
2400 61.950 Tetracosane 4.0
2500 64.577 Pentacosane 6.4
2600 67.090 Hexacosane 7.0
2649 68.119 10-Methyl hexacosane 2.1
2667 68.549 2-Methyl hexacosane 0.3
2700 69.506 Heptacosane 7.1
2768 70.913 2-Methy heptacosane 0.4
2778 71.147 3-Methyl heptacosane 0.3
2800 71.835 Octacosane 5.9
2834 72.433 Squalene 1.9
84
Table 3.12 (Continued)
2868 73.199 2-Methyl octacosane 0.5
2878 73.437 3-Methyl octacosane 0.2
2890 73.690 Cholesterilene 0.4
2900 74.083 Nonacosane 5.4
2924 74.448 12-Methyl nonacosane 1.2
2958 75.211 Unidentified 0.3
2968 75.409 2-Methyl nonacosane 0.3
2978 75.648 3-Methyl nonacosane 0.3
3000 76.252 Triacontane 3.5
3056 77.320 Unidentified 1.2
3067 77.544 2-Methyl triacontane 0.3
% Identified 98.0 93.9 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP 5ms column. b RT= Rention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization.
The major components of S. thurberi artificial rot are: hexanoic acid (69.6%), octanoic acid (9.9%), decanoic acid (4.0%), dodecanoic acid (3.4%), butanoic acid
(2.5%), and heptanoic acid (2.3%)(Table 3.13). The total carboxylic acid concentration for the lab rot is 91.7%. Previous studies have shown the toxic effects of hexanoic acid and octanoic acid on flies (100). Hexanoic acid was found to induce reversible coma in
Drosophila and octanoic acid to cause death (100). The extracts of both lab rots and the
85 early natural rot of S. thurberi were dominated by carboxylic acids, while carboxylic acids were minor components of the late field-rot specimens.
Table 3.13. Essential oil composition of lab-rot S. thurberi.
RIa RTb Compound % Lab Rot Stenocereus thurberi c
763 3.681 Butanoic acid 2.5
880 7.232 Pentanoic acid 0.1
967 9.421 Hexanoic acid 69.6
1074 20.495 Heptanoic acid 2.3
1167 20.548 Octanoic acid 9.9
1267 23.826 Nonanoic acid trd
1311 24.681 (2E,4E) Decadienal 0.1
1316 24.949 4-Methylpentyl hexanoate 0.4
1329 25.566 (2E,4E) Decadienol 0.1
1337 25.799 Methyl decanoate 0.1
1341 26.055 9-Decenoic acid trd
1362 26.911 2-Phenylethyl propanoate 0.1
1364 27.604 Decanoic acid 3.9
1375 27.127 Dihydro-5-pentylfuran-2(3H)-one trd
1377 27.517 dihydro-Eugenol trd
1444 30.323 2-Phenylethyl butanoate 0.4
86
Table 3.13 (Continued)
1450 30.580 2-Phenylethyl butyrate 0.4
1455 30.760 Phenyl octanoate trd
1458 30.899 (2E) Dodecanal trd
1461 31.022 (E)-5-Isopropyl-8-hydroxy-8-methyl-non-6-en-2- trd one
1468 31.301 Undecanoic acid trd
1478 31.668 γ-n-Heptylbutyrolactone 0.1
1487 32.081 2-Methyl-1-tetradecene 0.2
1499 32.612 Isovaleric acid trd
1503 32.710 Pentadecane 0.1
1513 33.135 2-Tridecanol 0.1
1521 33.421 Phenol, 2,4-bis(1,1-dimethylethyl-) 0.4
1526 33.851 Methyl dodecanoate 0.2
1537 34.038 Dihydroactinolide trd
1548 34.469 Phenylethyl valerate 0.1
1565 34.905 Dodecanoic acid 3.4
1616 37.135 Tetradecanal 0.1
1646 38.276 2-Phenylethyl hexanoate 0.2
1664 38.963 Tridecanoic acid 0.1
1684 39.627 1-Tetradecanol 0.3
1700 40.314 Heptadecane trd
1709 40.530 (2E)-Heptadecene trd
87
Table 3.13 (Continued)
1713 40.669 (2Z)-Heptadecene trd
1720 40.925 β-Santalol 0.1
1769 42.579 Benzyl benzoate trd
1769 42.713 Tetradecanoic acid 0.1
1781 43.580 Pentadecanol 0.1
1796 43.674 Ethyl tetradecanoate trd
1800 43.819 Octadecane 0.1
1815 44.314 n-Hexadecanol 0.1
1827 44.733 Isopropyl tetradecanoate trd
1845 45.275 i-Propyl 12-methyltetradecanoate 0.1
1853 45.397 Phenylethyl octanoate 0.1
1860 45.624 Benzoic acid, 2-phenylethyl ester 0.1
1865 45.787 Pentadecanoic acid trd
1866 45.944 3-Methyloctadecane trd
1879 46.375 14-Methyl pentadecanoate trd
1889 46.684 2-Methyl-1-octadecene 0.1
1897 46.934 1-Nonadecene 0.1
1904 47.161 Nonadecane trd
1911 47.528 2-Methyl-1-hexadecanol trd
1915 47.673 1,2,3,4-Tetrahydro-1,4-enthanoanthracen-2-one trd
1916 47.836 Unidentified 0.1
1922 48.046 Methyl Hexadecanoate 0.1
88
Table 3.13 (Continued)
1930 48.302 Cyclohexadecanolide trd
1962 49.036 Hexadecanoic acid 1.4
2000 50.264 Ethyl hexadecanoate 0.2
2000 50.387 Eicosane trd
2015 50.689 Hexadecyl ethanoate trd
2019 50.829 Unidentified trd
2026 51.184 Isopropyl hexadecanoate trd
2031 52.634 Palmitic acid, isopropyl ester trd
2079 53.245 Octadecanol trd
2092 53.245 cis-Linoleic acid 0.1
2098 53.437 8-Octadecenoic acid, methyl ester 0.1
2103 53.566 2-Methyl-Z-Z- 3,13-octadecadienol 0.1
2117 54.008 Lapachol 0.1
2141 54.177 Oleic acid trd
2169 54.410 Ethyl linoleate 0.2
2175 55.271 (Z,Z,Z)-9,12,15-Octadecatrien-1-ol 0.1
2195 55.458 Unidentified 0.1
2198 56.028 1-Docosene trd
2200 56.186 Docosane trd
2215 56.343 (E,E) 2,6,10-Nonadecatriene, 2,6,10,14-tetramethyl trd
2266 58.020 2-Phenylethyl laurate trd
2272 58.183 cis-10-Nonadecenoic acid trd
89
Table 3.13 (Continued)
2289 58.660 Pentadecyl hexanoate 0.1
2295 59.009 1-Tricosene trd
2300 59.161 Tricosane trd
2365 60.739 Eicosanoic acid trd
2384 61.396 Hexadecyl hexanoate trd
2396 61.722 1-Tetracosene trd
2400 61.856 Tetracosane trd
2496 64.313 1-Pentacosene trd
2500 64.459 Pentacosane trd
2597 66.829 1-Hexacosene trd
2600 66.963 Hexacosane trd
2698 69.262 1- Heptacosene trd
2703 69.373 Heptacosane trd
2770 70.974 Tetradecyl dodecanoate trd
2802 71.708 Octacosane trd
2831 72.366 Squalene trd
2900 73.967 Nonacosane trd
2971 75.475 Hexadecyl dodecanoate trd
3000 76.138 Triacontane trd
90
Table 3.13 (Continued)
3100 78.263 Untriacontane trd
% Identified 99.8
aRI=”Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5. bRT=Retention time on column. c The percentages of each component are reported as raw percentages based on total ion current without standardization. d tr = “trace” (< 0.05%).
The primary difference in composition between the late, naturally rotted S. thurberi specimens and the lab-rotted specimen is the percentage composition of carboxylic acids and p-cresol and p-methyl anisole, which are methyoxy phenol compounds. The natural late rot of S. thurberi is composed of 60.4% p-cresol and p- methyl anisole, while the lab rot is void of either of these two compounds. The dynamic headspace contained 33.3% p-cresol and p-methyl anisole. Although the lab rot is composed of 95.6% carboxylic acid, the natural rot is composed of just 3.8% carboxylic acid and 2.2% in the dynamic headspace extract. The lab rot was created during a 5 week time period. The lab rot had a green-brown, slushy appearance. The natural rot was dark brown in color with a thick consistency. Comparing the differences in the two specimens, it is likely that the lab rot simply was not given sufficient time to develop.
Additionally, the lab rot consisted of applying 6 microbes to the green cactus. The number and species of microbes on the field-rot was not ascertained. It is possible that the microbes colonizing the natural rot are more numerous and more diverse than that created in the lab. The field-rotted specimens should better reflect the natural conditions of the necrosis process.
91
3.5 Toxicity and Olfactory Preference of Drosophila melanogaster to cactus volatiles
The Drosophila Model proposes that the cactus fruit flies evolved from D. melanogaster. Toxicity tests were performed to determine the toxicity of certain compounds present in the cactus rots that are utilized by D. mojavensis, a cactiphilic fly inhabiting the Sonora Desert and Santa Catalina Island, California. A total of 16 compounds were tested for toxicity plus the green and necrotic forms of both O. littoralis and S. thurberi (Table 3.14). Of the compounds and specimens tested, benzaldehyde and phenylethyl alcohol were the most toxic to D. melanogaster (LC of 262 ± 35 µg/mL)
(Table 3.15). Of the three phenolic compounds tested, anisole was the most toxic with a
LC50 of 315 ± 35 µg/mL. Six carboxylic acids were tested: oleic acid, butanoic acid, palmitic acid, nonanoic acid, hexanoic acid, and octanoic acid. Nonanoic acid was the most toxic carboxylic acid tested. Both butanoic acid and palmitic acid were the least toxic carboxylic acids tested, and had a LC50 greater than 3000 µg/mL. The toxicity of the ketones tested varied in toxicity. 2-Heptanone was the most toxic ketone tested and isophorone was the least toxic. Two aldehydes were tested: benzaldehyde and pentanal.
Benzaldehyde had a LC50 fives times lower than that for pentanal.
Table 3.14. Toxicity of D. melanogaster to certain compounds identified in cactus rots.
Concentration % Lethality Standard LC50 (µg/mL) ppm ppm Deviation Ppm Phenol 393 ± 67
400 52.22 18.56
92
Table 3.14 (Continued)
200 15.00 8.37
Phenethyl alcohol 1954 ± 41
2000 50.00 0.00
1600 13.33 11.55
Benzaldehyde 262 ± 35
800 96.67 5.16
200 36.67 5.16
Nonanoic acid 365 ± 90
400 50.00 16.33
200 23.33 13.97
Hexanoic acid 2771 ±85
3000 50.00 0.00
1600 16.67 5.77
Octanoic acid 524 ± 36
800 100.00 0.00
400 13.33 11.55
2-Nonanone 419 ± 138
400 46.67 25.17
200 13.33 15.28
2-Heptanone 367 ± 90
1600 100.00 0.00
200 25.00 12.25
93
Table 3.14 (Continued)
Oleic acid 946 ± 313
1600 86.67 23.09
400 6.67 5.77
Pentanal 1423 ± 285
1600 60.00 20.00
800 13.33 5.77
Isophorone 1127 ± 300
1600 63.33 5.77
400 13.33 1.53
p-Cresol 458 ± 110
1600 100.00 0.00
200 13.33 13.66 p-methyl-Anisole 315 ± 30
400 83.33 15.28
200 0.00 0.00
Hexadecane 1279 ± 189
2000 53.33 5.77
800 46.67 5.77
Butanoic Acid >3000
Palmitic Acid >3000
94
The compounds tested for toxicity were chosen because of their presence in the necrotic cactus specimens. Although green specimens of O. littoralis were non-toxic to
D. melanogaster, the field-rot necrotic specimen was 100% lethal upon 48 hrs of exposure. Likewise, the early and late natural rots of S. thurberi were 100% lethal to D. melanogaster upon 48 hrs of exposure. The high concentrations of carboxylic acids, especially nonanoic acid and octanoic acid, in the lab and the early rot may account for the toxicity of the cacti specimens. The late natural rot is dominated by p-cresol, which is toxic (458 ± 110 µg/mL) to D. melanogaster.
Green O. littoralis does not contain any octanoic or nonanoic acid. But, necrotic
O. littoralis contains 8.8% octanoic acid and 0.1% nonanoic acid. Likewise, green S. thurberi lacks both octanoic acid and nonanoic acid. But, early natural rot S. thurberi is composed of 15.6% octanoic acid and 11.1% nonanoic acid. The lab-rotted S. thurberi is composed of 9.9% octanoic acid and a trace of nonanoic acid. Octanoic acid and nonanoic acid may lead to the death of D. melanogaster when grown on necrotic cactus.
p-Cresol was only present in the late, natural-rot S. thurberi. p-Cresol constitutes
55.9% of the late natural rot of S. thurberi. The late, natural rot is void of octanoic acid or nonanoic acid. This rot also contains 2-heptanone (0.4%) and p-methyl-anisole
(4.5%), which are toxic to D. melanogster (2-heptanone: 367 ± 90 µg/mL/ p-methyl- anisole: 315 ±30 µg/mL). The high concentration of toxic p-cresol in combination with the presence of 2-heptanone and p-methyl-anisole may account for the toxicity of late necrotic, field rot of S. thurberi.
Benzaldehye is toxic to D. melanogaster. But benzaldehyde is present in green O. littoralis (0.5%) as well as early (0.1%) and late rot S. thurberi (0.5%). Due to the
95 presence of benzaldehyde in the green, non-toxic O. littoralis, it is doubtful that this compound accounts for the toxicity of the S. thurberi rots to D. melanogaster.
LC50 toxicity numbers were found to be greater than 3000 µ/mL for both palmitic acid and butanoic acid. Palmitic acid is found in green O. littoralis (4.4%), lab-rot O. littoralis (0.8%), late field-rot S. thurberi (2.5%), and lab rot S. thurberi (1.4%). The higher concentraction level of palmitic acid in the non-toxic green O. littoralis indicates that this compound probably is not responsible for the toxicity of the necrotic cacti.
Butanoic acid is present in the lab rot of O. littoralis (1.0%), early natural rot S. thurberi
(33.0%), and lab rot S. thurberi (2.5%). Because butanoic acid has a high LC50, it is probably not responsible for the lethality of the necrotic cacti.
Phenol is found in both the early S. thurberi rot (0.4%) and the dynamic headspace of the late rot (0.3%). Phenol may account for some of the toxicity of necrotic
S. thurberi, but not the late rot, which is void of phenol.
Olfactory preference testing with D. melanogster did not reveal a statistical preference for any of the compounds or cactus specimens tested. A lack of preference may be due to the lack of any of these compounds or specimens being within the native diet of D. melanogaster.
96
3.6 Characterization of Crystals and Silica Bodies Isolated from O. littoralis and Stenocereus thurberi.
Microscopic examination of cut cactus specimens revealed crystal bodies throughout the biomass of both O. littoralis and S. thurberi. This is the first identification of crystals in an Opuntia species. Crystals (Figures 3.6 and 3.7) identified in O. littoralis and O. ficus-indica had a starburst shape identical to the crystals identified as silica bodies in micrographs published by Gibson and Horak in 1978 (32). Crystals identified in S. thurberi (Figures 3.8 and 3.9) had a rod shape, which differs from the starburst shape identified in columnar cactus specimens investigated by Gibson and Horak (32).
Smaller pads of O. littoralis and O. ficus-indica were laden with small crystals (~50 microns) while larger crystals were identified in larger cactus pads. Although prior research identified crystal bodies in the epidermis of columnar cactus (32), no crystals were found in the cactus dermis of either species examined for this study. Previous work established that these crystals are composed of silica (32).
97
Figure 3.8. O. littoralis magnified at 100X. Figure 3.9. Crystal removed from O. littoralis.
Figure 3.10. S. thurberi magnified at 100X. Note Figure 3.11. Crystal removed from S. thurberi. crystals are white rod-shapes embedded in biomass.
Silica is composed of a tetrahedral structure wherein a silicon atom is bond to 4 oxygen atoms (101). Each oxygen atom is bound to 2 silicon atoms (101). The O-Si-O bond within silica has a bond angle of 109°, which prevents rotation about the silicon atom without the input of energy (101). The oxygen bond within silica, Si-O-Si, freely
98 rotates from 130° to 180° withough the addition of energy (101). The Si-O-Si bond flexibility allows silica polymorphs to exists that range from dense crystals to aerogels
(101). These silica polymorphs have differing chemical, physical, and structural properties (101). Although silica does not contain any hydrogen, it can be analyzed by
1H NMR because it rapidly protonates from humidity in the air that contacts the sample
(102). This process is called physiosorption. The speed of water physiosorption at room temperature with silica is dependent of the silica ring size (102). Silica also rapidly protonates in the presence of an alcohol such as methanol, which opens the strained silicon-oxygen ring (102). This protonation creates a silanol on the surface of the silica crystal that can be identified with 1H NMR (102). Two main silanol peaks are observed
(102). The first peak is observed at chemical shifts near 1.61 ppm (102) and 1.7 ppm
(103), which is assigned to non-interacting silanols (102). These non-interacting silanols are considered to be isolated (103) silanol groups. The second, down-field peak or peaks is observed at a chemical shift between 3.0 ppm and 6.5 ppm (102) (103). The second peak is the Si-OH with the highest affinity for water (102). This downfield peak is a well-defined peak that shows the distribution of hydrogen-bonded hydroxyl groups (103).
1H NMR, and 13C NMR (nuclear magnetic resonance spectroscopy) testing was performed on the crystal particulate extracted from O. littoralis and S. thurberi during dynamic headspace extraction, which produced in excess of 100 mg of crystalline particulate for both cacti species, to determine whether the crystals were composed of silica or cyclic siloxane compounds.
99
The results for O. littoralis are 1H NMR (500 MHz, Chloroform-d) δ 4.46 (s,
13 1H), δ 2.21 (d, 2.64 9H, 1.80 6H) ; and C NMR (CDCl3) had a faint peak at δ 32.37(s).
The proton at δ4.46 is a well-defined peak that corresponds to the sight with the highest affinity for water. The proton doublet at chemical shift δ 2.21 ppm corresponds to an isolated silanol function.
Nuclear magnetic resonance spectroscopy analysis on S. thurberi Engelm. produced the following results: 1H NMR (500 MHz, Chloroform-d) δ5.23 (s, 1H), δ
13 1.33 (d, J=151.3Hz, 23H), and a faint C NMR (CDCl3) peak at δ 32.41.
The nuclear magnetic resonance results are consistent with the crystalline particulate being silica. Both cactus species produced 1H NMR spectra with two peaks.
The downfield peaks at chemical shifts of 4.46 and 5.23 ppm correspond to silanol peaks with high affinity for water. Both upfield peaks, which were at chemical shifts of 2.21 ppm and 1.33 ppm, are consistent to the upfield, non-interacting silanol peaks. Although the actual values for the chemical shifts are different from the published values, more than 200 polymorphs of slica are theoretically possible, and the structure of the crystals herein has not been determined. Additionally, the upfield peaks were split for both cactus crystalline extracts. This indicates that there are multiple silanol sights on the surface of the silica polymorphs.
Although 13C NMR was able to identify a small carbon peak for both cactus specimens, carbon from the solvent is known to interact with silanol groups in 13C NMR spectra (104) (103). The small carbon peaks may be the interaction of a carbon atom from the solvent with the upfield, non-interacting silanol peak.
100
The crystalline particular could not be cyclic siloxanes, because cyclic siloxanes are neat liquids. Although linear siloxanes have proton chemical shifts less than 1.0 ppm, there are no published values for cyclic siloxanes. Carbon nuclear magnetic resonance chemical shifts for linear polydimethylsiloxane are negative values and chemical shifts for cyclic polydimethylsiloxanes are in the range of 0 to 1.5 ppm (105).
Specimens of O.littoralis, S. thurberi, and O. ficus-indica were stained with
PDMPO and observed for fluorescence. The specimens were viewed with the Nikon compound microscope wit h a fluorescent attachment (Ex: 333-385 nm; Em: 400-700 nm). There was no visible fluorescence. The specimens were also viewed under the confocal microscope at Ex: 419 nm, which was the lowest level available on the Zeiss confocal microscope. Although PDMPO fluoresces at excitation of 333-385 nm, that level of excitation was unavailable on the confocal microscope. None of the crystals identified in either cactus specimen had a positive fluorescence (Figures 3.10-3.13).
Additionally, there was an absence of silica fluorescence in any of the specimens studied.
101
Figure 3.12. O. littoralis specimen stained with Figure 3.13. S. thurberi specimen stained with PDMPO. 630X magnification. PDMPO. 100X magnification.
Figure 3.14. O. ficus-indica crystal body within Figure 3.15. O. ficus-indica stained with PDMPO. the biomass of the cactus. Magnification 100X. Note the lack of staining of crystal body. Magfification 100X.
102
Three samples from six different plants of O. littoralis, S. thurberi (Figure 3.14), and O. ficus-indica were tested for the presence of cyclic siloxanes using GC-MS. All of the O. ficus-indica and S. thurberi plants tested positive for cyclic polydimethylsiloxane
(Tables 3.14 and 3.15). Five of the six O. littoralis plants tested positive for cyclic polydimethylsiloxane (Table 3.14). The cyclic oligosiloxanes identified from GC-MS analysis of the hydrodistillation extracts are not included in the preceeding tables that list the essential oil composition of the cactus species because cyclic oligosiloxane is not an essential oil. The percentage composition listed for each oil component in the preceeding tables is the percentage that oil component is of the total essential oil composition, which excludes cyclic oligosiloxane compounds.
Figure 3.16. GC-MS spectra of green S. thurberi. Cyclic polydimethylsiloxane peaks are at 44.983, 50.021, 54.636, 58.853, and 62.757 minutes.
103
Table 3.15. Oil recovered from hydrodistillation of green Opuntia cacti specimens and presence of cyclic oligosiloxane compounds.
Cactus Species (Sample) Specimen (g) Extract Cyclic Recovered (mg) Oligosiloxane presence confirmed by GC- MS O. littoralis
Green (Matzkin Sample) 144.91 4.7 +
O. littoralis
1A 145.51 0.6 +
1B 158.63 125.2 +
1C 159.62 443.3 +
2A 162.74 1.2 +
2B 159.08 679.3 +
2C 153.30 0.8 +
3A 151.99 5.8 -
3B 122.33 209.3 -
3C 156.24 3.0 -
4A 164.37 0.5 +
4B 149.08 3.7 +
4C 158.30 85.8 +
5A 146.28 10.1 +
5B 140.85 1.9 +
104
Table 3.15 (Continued)
5C 162.18 4.4 +
6A 154.17 2.6 +
6B 164.28 823.7 +
6C 121.41 517.9 +
O. ficus-indica
1A 172.56 2.2 +
1B 195.86 3.0 +
1C 150.28 13.8 +
2A 147.67 2.8 +
2B 148.30 21.0 +
2C 151.80 1.5 +
3A 166.16 4.9 +
3B 146.43 133.0 +
3C 150.33 14.6 +
4A 152.65 183.6 +
4B 155.74 87.0 +
4C 149.00 3.1 +
5A 162.18 259.8 +
5B 160.32 32.9 +
5C 150.16 12.8 +
6A 157.30 4.5 +
105
Table 3.15 (Continued)
6B 162.69 1.9 +
6C 153.28 3.0 +
Table 3.16. Oil recovered from hydrodistillation of necrotic O. littoralis, green S. thurberi, and necrotic S. thurberi and presence of cyclic oligosiloxane compounds.
Cactus Species and # Specimen (g) Extract Cyclic Recovered (mg) Oligosiloxane presence confirmed by GC-MS O. littoralis
Lab rot 48.3 1.1 +
O. littoralis natural rot +
Dynamic headspace 1,478 949.8 tra
Rot 1A 201.14 0.5 +
Rot 1B 198.46 0.8 +
Rot 1C 193.78 1.4 +
Rot 1D 199.86 1.3 +
S. thurberi
Green 121.51 1.3 +
S. thurberi
Lab rot 43.63 1.1 +
106
Table 3.16 (Continued)
S. thurberi natural rot
Dynamic headspace 4,869 328.60 tra
Rot 1Ab 162.08 0.9 +
Rot 1Bb 169.14 1.1 +
Rot 2Ab 163.23 1.3 +
Rot 2Bb 173.98 35.0 +
Rot 3Ab 150.71 0.8 +
Rot 3Bb 149.51 0.6 +
Rot 4A 163.12 91.9 +
Rot 5A 174.81 52.7 +
Rot 6A 46.01 36 +
Rot 6B 51.31 29 +
atr = “trace” b=Early necrotic state
The percentage of the total extract composed of PDMS varied greatly among the cacti specimens tested. The total distillation extract of green S. thurberi (distillate yield of 1.07 x 10-1 %) was composed of 81.0% PDMS compounds and the lab rot distillate of
S. thurberi (distillate yield of 2.52 x 10-3 %) was composed of 5.1% PDMS compounds.
The total distillate extract of green O. littoralis (distillate yield of 3.24 x 10-3%) contained
51.3% PDMS compounds, but the lab rot distillate of O. littoralis (distillate yield of 2.77 x 10-1%) was composed of just 4.7% PDMS compounds.
107
Five different cyclic oligosiloxanes were identified in O. littoralis (Table 3.16): hexadecamethyl-cyclooctasiloxane, octadecamethyl-cyclononasiloxane, eicosamethyl- cyclodecasiloxane, 2,4,6-trimethyl-2,4,6-triphenyl-cyclotrisiloxane, and tetracosamethyl- cyclododecasiloxane. Three cyclic polydimethylsiloxane compounds were identified in green S. thurberi (Table 3.16): octadecamethyl-cyclononasiloxane, eicosamethyl- cyclodecasiloxane, and tetracosamethyl-cyclododecasiloxane. And, three cyclic polydimethylsiloxane compounds were identified in green O. ficus-indica (Table 3.16): hexadecamethyl-cyclooctasiloxane, octadecamethyl-cyclononasiloxane, and eicosamethyl-cyclodecasiloxane. The percentage composition shown for each compound in Table 3.18 is the percentage of all PDMS compounds contained in the extract. Essential oil components are excluded in the computation of the percentage composition of each PDMS compound.
Table 3.17. Volatile cyclic siloxane composition of green specimens of O. littoralis, S. thurberi, and O. ficus-indica.
RIa RTb Compound % green % green % green O. S. O. ficus- littoralisc thurberic indicac
39.348 Cyclooctasiloxane, 3.4 1674 hexadecamethyl- 8.6
1774 42.969 Unidentified Cyclic Siloxanec 0.8 1.1
44.984 Cylcononasiloxane, 25.5 21.1 1835 octadecamethyl- 22.7
1992 50.020 Cyclodecasiloxane, eicosamethyl- 26.0 31.8 49.8
108
Table 3.17 (Continued)
2139 54.637 Unidentified Cyclic Siloxaned 23.9 24.5 25.7
2153 55.091 Unidentified Cyclic Siloxaned 0.2
2186 55.772 Unidentified Cyclic Siloxaned 0.3
2245 57.443 Unidentified Cyclic Siloxaned 0.2
2258 57.810 Unidentified Cyclic Siloxaned 0.2
2295 58.852 Unidentified Cyclic Siloxaned 11.6
59.912 Cyclotrisiloxane, 2,4,6-trimethyl- 2328 2,4,6-triphenyl- 1.2
62.759 Tetracosamethyl- 14.5 2430 cyclododecasiloxane 3.4
2579 66.427 Unidentified Cyclic Siloxaned 1.2 1.0
2723 69.880 Unidentified Cyclic Siloxaned 0.4 0.3
2864 73.105 Unidentified Cyclic Siloxaned 0.2
3001 76.138 Unidentified Cyclic Siloxaned 0.1 0.2
% Identified 61.9 71.8 74.3 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT= Retention Time on column c The percentages of each component are reported as raw percentages based on total ion current without standardization. d MS, m/z(%): : 73.1, 147.1, 221.1, 281.1, and 355.1. Bold=% Identified in bold are primary components.
There are no published retention index values for cyclic oligosiloxanes larger than tetracosamethyl-cyclododecasiloxane. But, cyclic oligosiloxane compounds larger than hexadecamethyl-cyclooctasiloxane have the same GC-MS spectra with
109 fragmentation at 73.1, 147.1, 221.1, 281.1, and 355.1 m/z (Figure 3.15) (106). This fragmentation pattern was seen in the spectra for green S. thurberi (Figure 3.15)
Figure 3.17. GC-MS spectra of peak 44.955 minutes from green S. thurberi. Note m/z: 73.1, 147.1, 221.1, 281.1, 355.1, 429.1 are characteristic of polydimethylsiloxane. The peak at m/z of 73.1 is trimethylsilyl (the characteristic fragment of trimethylsilyl ion). m/z 281.1 is cyclooctasiloxane, hexadecamethyl-.
The plant lacking cyclic siloxane on GC-MS was found to be lacking crystal bodies during microscopic investigation. The O. littoralis specimen lacking crystals
(Table 3.17) and the specimen with crystals (Table 3.1) had a number of oil components common to both specimens. 64 compounds were identified in the extract for the cactus lacking crystals. The volatile extract from O. littoralis lacking silica bodies is composed
110 of 44.1% terpene compounds, 19.1% aldehyde-derived compounds, 5.4% furanoid and benzene compounds, 5.1% alkane compounds, and just 0.4% fatty acid. O. littoralis with crystals contained 47.2% terpenoid-derived compounds, 29.1% fatty acids, 16.2% aldehydes, 7.6% alkanes, and 10.6% furanoid and benzenoid compounds. The most significant change in composition is the decrease in fatty-acid-derived compounds in the non-silica cactus. The terpenoids, aldehydes, alkane, furanoid, and benzene concentrations were similar in both cactus specimens.
The linalool concentrations of both the crystal-laden and non-crystal cactus plants were similar. The essential from O. littoralis lacking crystals is composed of 22.0% linalool compounds (cis-linalool oxide, 10.9%, trans-linalool oxide 8.9%, linalool 1.8%, and (E,E) geranyl linalool 0.4%) while O. littoralis without silica bodies contains 21.3% linalool compounds (cis-linalool oxide 10.8%, trans-linalool oxide 8.8%, and linalool
1.7%).
Compounds identified in the non-siloxane sample, but not in the siloxane-laden cactus, include piperonal (3.0%), aromadendrene (2.4%), α-calacorene (3.1%), patchoulane (0.6%), muurola-4,10 (14)dien-1-β-ol (4.1%), ledene oxide-(II) (2.8%), longiverbenone (0.8), cis-calamenen-10-ol (2.1%), trans-calamenen-10-ol (0.9), naphthalene, 1,6-dimethyl-4-(1-methylethyl)- (1.1%), aromadendrene oxide-(2) (1.6%), cis-Z-α-bisabolene epoxide (0.6%), 5,6,6-trimethyl-5-(3-oxobut-1-enyl)-1- oxaspiro[2.5]octan-4-one (1.3%), and 2-propenal, 3-(4-hydroxy-3-methoxyphenyl)-
(2.3%). These compounds account for 26.7% of the total non-crystal O. littoralis essential oil. 18.2% of the essential oil of the non-crystal specimen could not be identified.
111
Compounds found in the crystal-rich cactus, but not the O. littoralis void of crystals, include santene (0.3%), p-cymene (1.4%), phenylacetaldehyde (1.4%), g- terpineol (0.9%), p-menth-1-en-9-al (cis and trans)(1.2%), β-cyclocitral (0.6%), vitispirane (1.0%), (3S,6S,7E)-7,10-epoxy-2,6,10-trimethyl-2,5,11-dodecatriene (cis and trans) (4.8%), α-cadinol (0.4%), and (E)-phytol (1.2%).
Table 3.18. Volatile composition of Opuntia littoralis lacking crystals.
RIa RTb Compound % Opuntia littoralis without silica bodiesc
796 3.500 Hexanal 4.7
825 4.085 Propane, 2,2'-[ethylidenebis(oxy)]bis- 0.6
834 4.272 Furfural 2.2
870 5.147 Hexanol 0.3
888 5.772 Unidentified 0.2
903 6.203 Heptanal 0.4
907 6.450 Butyl propanoate 0.3
932 7.012 Ethanone, 1-cyclopentyl- 0.1
959 8.261 Heptenal <(2E)-> 0.3
964 8.459 Benzaldehyde 0.4
966 8.563 Hepten-1-ol <(4Z)-> 0.3
981 9.254 Dimethyl-4-heptanone <3,5-> 0.1
112
Table 3.18 (Continued)
988 9.604 Hepten-2-one <6-methyl-5-> 0.3
1003 10.294 Octanal 0.2
1016 10.874 2,4-Nonadiene, (E,E)- 0.2
1032 11.705 Cyclohexanone, 2,2,6-trimethyl- 0.3
1058 12.838 Octen-1-al <(2E)-> 0.2
1065 13.178 Acetophenone 0.3
1072 13.515 Linalool oxide
1087 14.261 Linalool oxide
1094 14.563 Benzoic acid, methyl ester 0.2
1100 14.819 Linalool 1.8
1104 15.033 Nonanal 1.7
1149 17.152 Ethanone, 1-(1,4-dimethyl-3-cyclohexen-1-yl)- 0.2
1159 17.662 2-Nonenal, (E)- 0.3
1175 18.427 Unidentified 0.4
1184 18.845 Cymen-8-ol
0.7
1189 19.087 Terpineol <α-> 0.7
1193 19.280 Methyl salicylate 1.0
1205 19.842 Decanal 0.2
1213 20.237 3-Cyclohexene-1-acetaldehyde, α,4-dimethyl- 2.0
1220 20.599 Unidentified 0.5
1225 20.841 Sabinene hydrate acetate
1374 27.424 Copaene <α-> 0.4
113
Table 3.18 (Continued)
1397 28.395 1,3-Diethyladamantane 0.2
1400 28.501 Tetradecane 0.4
1409 28.882 Dodecanal 0.4
1438 30.075 Aromadendrene 2.4
1453 30.708 Geranyl acetone 0.4
1460 30.976 Aromadendrene
1462 31.080 Tetradecane, 4-methyl- 0.3
1477 31.657 Cadina-1-(6),4-diene
3-Buten-2-one, 4-(2,5,5-trimethyl-3-oxatricyclo 1486 32.047 [5.1.0.0 (2,4)]oct-4-yl)-, [1α,2α,4α(E),7α]- 0.5
1500 32.621 Pentadecane 1.0
1514 33.161 Cadinene <γ-> 0.3
1543 34.295 α-Calacorene 3.1
1557 34.850 Unidentified 0.3
1578 35.743 Diethyltoluamide 0.6
1587 36.027 Copaen-4-α-ol <β-> 0.5
Propanoic acid, 2-methyl-, 1-(1,1- 1598 36.452 dimethylethyl)-2-methyl-1,3-propanediyl ester 0.3
1600 36.518 Hexadecane 0.7
1615 37.127 Patchoulane 0.6
1628 37.592 Muurola-4,10(14)dien-1-β-ol 4.1
1642 38.093 Ledene oxide-(II) 2.8
1654 38.556 Longiverbenone 0.8
114
Table 3.18 (Continued)
1658 38.737 Calamenen-10-ol
1666 39.068 Calamenen-10-ol
1673 39.301 Naphthalene, 1,6-dimethyl-4-(1-methylethyl)- 1.1
1681 39.536 Aromadendrene oxide-(2) 1.6
1700 40.221 Unidentified 1.8
1710 40.570 cis-Z-α-Bisabolene epoxide 0.6
5,6,6-Trimethyl-5-(3oxobut-1-enyl)-1- 1736 41.550 oxaspiro[2.5]octan-4-one 1.3
1760 42.228 2-Propenal,3-(4-hydroxy-3-methoxyphenyl)- 2.3
1765 42.588 Unidentified 1.6
1770 42.763 Unidentified 0.9
1777 43.074 Unidentified 4.0
1780 43.179 Unidentified 1.8
1786 43.387 Unidentified 1.1
1789 43.479 Muurolene <14-hydroxy-α-> 1.3
1811 44.195 Unidentified 1.5
1844 45.279 Unidentified 1.4
1881 46.452 Unidentified 1.0
1946 48.822 Unidentified 0.4
1996 50.129 Unidentified 0.3
2001 50.280 Eicosane 0.2
2028 51.113 Hexadecanoic acid, 1-methylethyl ester 0.4
115
Table 3.18 (Continued)
2198 56.284 Docosane 0.3
2298 59.099 Tricosane 0.5
2354 60.616 Unidentified 0.2
2368 60.980 Unidentified 0.9
2801 71.675 Nonacosane 1.4
2829 72.312 Squalene 1.6
2900 73.909 Nonacosane 0.3
% Identified 81.8 a RI = “Retention Index” determined in reference to a homologous series of n-alkanes on an HP-5ms column. bRT= Retention Time on column c The percentages of each component are reported as raw percentages based on total ion current without standardization. Bold=% Identified in bold are primary components.
The amount of cyclic siloxane polymers recovered from the cactus specimens were so small that additional analysis was not feasible. The only analysis that could be performed on the extracts containing cyclic siloxanes was GC-MS, which requires minute amounts of oil. The quantities of extract obtained from hydrodistillation of the cactus specimens was insufficient to characterize via nuclear magnetic resonance spectroscopy,
Raman spectroscopy, or UV/Vis spectrophotometry.
X-ray diffraction analysis was unable to characterize the crystal structure of the silica bodies dissected from O. littoralis, O. ficus-indica, and S. thurberi. The crystals dissected from the S. thurberi specimens were 50 microns or less in diameter. These
116 crystals were too small to be analyzed by the x-ray diffraction instrument employed herein. The crystals dissected from both O. littoralis and O. ficus-indica were at least 70 microns in diameter, which is large enough for the x-ray diffraction analysis performed herein. But the Opuntia crystals in excess of 70 microns in diameter were not single crystals and could not be analyzed. Unfortunately, the structure of the silica crystals could not be identified in this study.
117
CHAPTER 4
DISCUSSION
This chapter discusses the results obtained in this study. This is the first study of the essential oil composition of the following species of American cacti: Opuntia littoralis (Engelm.) Cockerel, Opuntia ficus-indica (L.) Mill, and Opuntia prolifera
Engelm., Opuntia acanthocarpa var. major and Opuntia phaeacantha var. discata, and
Stenocereus thurberi Engelm. Quantities of oil extracted were too small for natural product testing. But, the complex and diverse nature of the oil content and the sequester of silica in crystal bodies within the biomass reflects the environmental challenges each cactus species has successfully adapted to.
4.1 Santa Catalina Island, California Opuntia Species
This study investigated three Opunita species growing on Santa Catalina Island,
California: Opuntia littoralis (Engelm.) Cockerel, Opuntia ficus-indica (L.) Mill, and
Opuntia prolifera Engelm. Each of these three species had differing chemical
118 compositions. O. littoralis was dominated by terpenoid-derived compounds (47.2%), the
O. ficus-india was composed primarily of fatty acids (49.7%), and O. prolifera was dominated by alkanes (47.1%). None of these three plants shared any major components in similar percentages of composition. This is the first study of the essential oils of these
Santa Catalina Island, California Opuntias.
4.2 Southern Arizona Opuntia Species
This study characterized the essential oil composition of two Opuntia species-
Opuntia acanthocarpa var. major and Opuntia phaeacantha var. discata, growing native in the loamy soil of the Organ Pipe Cactus National Monument and the caliche soil of the
Arizona-Sonora Desert Museum. The findings show that the phenotype of the two cactus species adapts to the environmental conditions of the two differing areas of the Sonora
Desert. Opuntia specimens growing at the Organ Pipe Cactus National Monument had lower essential oil concentrations than the same species growing at the Arizona-Sonora
Desert Museum. The Arizona-Sonora Desert Museum has a greater amount of annual precipitation than the Organ Pipe Cactus National Monument and has a high level of caliche soil, which is absent at the Organ Pipe Cactus National Monument. Higher levels of rainfall and/or higher levels of caliche may induce higher oil production in these two species. Additionally, the alkane concentrations of both cactus species was greater in the
Organ Pipe Cactus National Monument specimens than the Arizona-Sonora Desert specimens. The drier, loamy soil of the Organ Pipe Cactus National Monument may induce alkane formation in these Opuntia species.
119
4.3 Stenocereus thurberi Engelm. growing at Organ Pipe Cactus National Monument
This study characterized the volatile fraction of S. thurberi native to the Organ
Pipe Cactus National Monument. This is the first characterization of this unique
American cactus. The essential oil composition of S. thurberi varied significantly from the Opuntia species studied in this dissertation. Organ pipe cactus contained the highest level of alkene (27.5%) concentration of any cactus studied herein. Although the large, older pads of Opuntia obtain a wood appearance, S. thurberi contains a porous, wood inner skeleton. Lapachol, a napthoquinone found thus far only in Central and South
American trees, was identified in the essential oil of organ pipe cactus.
4.4 Necrotic Specimens of O. littoralis and S. thurberi
O. littoralis and S. thurberi are utilized by the cactus fly Drosophila mojavensis.
These cactus rots are toxic to Drosophila melanogaster, the fruit fly. This study investigated the essential oil composition of lab-rotted and field-rotted specimens.
Essential oil compositions and dynamic headspace extractions of O. littoralis for both dynamic headspace and rot were unsuccessful. Only trace amounts of oil could be extracted. The green specimen of O. littoralis contained sufficient oil to characterize its composition. The major difference in the field-rot and the lab-rot specimens are the appearance of the cactus and the percentage of hydration. The lab rot specimen is green/brown in color and is full of liquid. The natural rot is dry. The outside of the cactus pad obtains a bark-like appearance and the flesh of the pad turns while/yellow
120 until it reaches the dark brown of full necrosis. The microbes used to produce the lab rot may affect the drying process of the cactus pads.
The early field rot and lab-rotted specimens of S. thurberi had similar total carboxylic acid concentrations, 84.8% and 91.7% respectively. The late rot of S. thurberi was markedly different in essential oil composition. The late rot was dominated by p- cresol. The field rots are allowed to mature for 1 to 9 weeks. The S. thurberi field rots were in the field for approximately six months rotting. It may be that the lab rots were terminated prior to the completion of the rot.
Compounds were identified in each of the necrotic cactus specimens that are toxic to D. melanogaster. Small concentrations of essential oil were identified from the lab rots of both cactus and the early rot of S. thurberi. Larger quantities of oil were extracted from the necrotic specimens of S. thurberi late rot than either the lab-rot or early rot specimens. Despite the difference in percentage of oil composition, both the early and late rots were lethal to the fruit flies upon 48 hours of exposure.
The lab rot of O. littoralis and the early and lab rots of S. thurberi are dominated by carboxylic acids. Octanoic and nonanoic acid were found to be highly toxic to the fruit flies. These two compounds probably account for the majority of the toxicity of fruit flies to lab rot O. littoralis and early natural and lab rot S. thurberi.
The late rot of S. thurberi lacked the high carboxylic acid concentrations that were identified in the lab rot of O. littoralis and early, natural and lab rots of S. thurberi.
Instead, the late, natural rot of S. thurberi was dominated by p-cresol. Both p-cresol and p-methy-anisole, which are identified herein as being toxic to D. melanogaster, were
121 present in the late, natural rot of S. thurberi in signicant concentrations. The high concentration of both p-cresol and p-methyl-anisole in the late, natural rot may account for the toxicity of cactus to fruit flies.
4.5 Characterization of cyclic siloxane compounds in O. littoralis, O. ficus-indica, and S. thurberi
Crystal bodies were identified in O. littoralis, O. ficus-indica, and S. thurberi.
The crystal bodies in both Opuntias had a starburst shape while the crystals in S. thurberi had a rod shape. No crystals were identified in the epidermis of any of the species studied herein. This is the first identification of cystal bodies in an Opuntia species. The crystals have previously been identified as silca bodies. The nuclear magnetic resonance analysis corresponds to the characterization of the crystals as being composed of silicon dioxide.
The crystal bodies could not be characterized by fluorescent microscopy. None of the cactus specimens had a positive fluorescence for silica using the PDMPO stain. GC-
MS was used to characterize the volatile cyclic siloxane components of the extracts of each of the three cacti species. GC-MS analysis suggests that each cactus species contains large, cyclic oligosiloxanes.
Cyclic oligosiloxanes are incorporated into health care and beauty products readily available over the counter. Cyclic oligosiloxanes are also contaminating by- products of the production of linear polydimethylsiloxanes, which have a number of
122 industrial applications including mechanical grease. Large, cyclic oligosiloxanes have not been studied independently.
A major concern is whether the polydimethylsiloxanes identified in this study could be a contaminat of silicone grease commonly used in chemistry labs. No grease was used in any of the hydrodistillation equipment utilized herein. Extractions were performed numerous times over a period of approximately two years. Additionally, extractions were performed on other plant species during this time period and none of them contained any polydimethylsiloxane compounds.
An additional concern is whether the polydimethylsiloxanes could be a contaminating by-product of the GC-MS column. Oligosiloxanes are known to bleed from the column and be seen on GC-MS spectra. But, the percentage of oligosiloxanes that bleed off the column is very small, and here the polydimethylsiloxane compounds were a majority of the volatiles in some of the extractions performed. Addtionally, the experimental conditions utilized on the GC-MS reach temperatures sufficient high enough to elute oligosiloxanes from the silica column only at temperatures reached during the last few minutes of the run, 70 minutes or later. During the processing of the extractions in this study, polydimethylsilxoanes eluted from the GC-MS column as early as 35 minutes after the run was begun. Column bleed does not typically occur at a retention time of 35 minutes. Finally, column bleed does not typically form discrete peaks. Column bleed is seen when the baseline rises (107). The discrete peaks seen in this study do not match typical column bleed (Figure 4.1).
123
Figure 4.1. Figure printed from Agilent Technologies. (107). Example of column bleed.
Septum bleed can not be an issue in this study because a degrading septum would bleed in all samples until replaced. Of all samples run on the GC-MS used in this study only the cactus samples listed in Tables 2.1 and 2.3, above, had cyclic polydimethylsiloxane peaks.
Whether the cyclic oligosiloxane compounds identified in this study are a by- product of the distillation of the silica bodies contained within the cactus must be considered. Polydimethylsiloxane is not a by-product of silica formation or hydrodistillation in the laboratory. It is unlikely that the polydimethylsiloxanes identified in the GC-MS spectra in this study are contaminants from the extraction procedures utilized in this study.
Water and alcohols interact with the surface of silica (108) (101), open the siloxane ring, and produce silanols (101). The process of silca interacting with water or liquids is called physiosorption. It is possible that the cyclic oligosiloxanes identified in the cacti herein are the result of defects that occur during physiosorption. The process of
124 ring opening with water and methanol physiosorption has been found to cause defects in the silica transforming the silica so that additional moieties such as hydroxyl groups are added to the silicon dioxide . Physiosorption may lead to the formation of polymethylsiloxanes within the cactus, a plant containing water and a variety of oils.
There is such a large concentration of silica bodies within all three species investigated:
O. littoralis, S. thurberi, and O. ficus-indica. It is plausible that the cyclic oligosiloxanes identified in GC-MS are formed from physiosorption.
The plant lacking cyclic siloxanes also lacked silica bodies. This suggests that the sequester of silicic acid within the cactus in the form of silica, silicon dioxide, is related to the production of cyclic oligosiloxanes. An enzymatic process is necessary for a cactus to sequester silicon. The formation of the cyclic siloxanes may be a by-product of the enzymatic conversion of silicic acid into silica. There are such large quantities of silica within the cactus, and such small quantities of cyclic siloxane that the formation of cyclic siloxanes as a contaminant in the production of silica crystals is a plausible explanation.
125
CHAPTER 5
CONCLUSION
5.1 Findings
The cacti species of the North American deserts have a varied and diverse composition. Species within the same genus have vastly different volatile compositions.
The same species when confronting different climate and soil undergo phenotype changes in volatile composition. Some cactus species such as S. thurberi may live for hundreds of years in an arid, inhospital climate. How plants adapt to this severe climate may lead to a better understanding of how other non-cactus species may respond to future changes in climate.
Opuntia acanthocarpa var. major (Engelm. & J.M. Bigelow) L.D. Benson and
Opuntia phaeacantha var. discata (Griffiths) L.d. Benson & Walk are two species growing in the Sonora Desert of Arizona that are able to adapt and thrive in different climes and soil. This adaption to environment includes differing volatile compostions.
126
Cactus flies feed off of the necrotic rots of Opuntia littoralis and Stenocereus thurberi Engelm., which are members of two different families of cactus. The rots of these cacti are complex and intriguing. Fruit flies are unable to utilize these rots as a food source. This implies that the cactus flies have evolved over time in order to be able to utilize these rots that are lethal to the fruit flies.
During the 1970’s, crystals were identified in the cactoid family of the cactus species. These crystals were determined to be silica bodies. Although the crystals studied herein did not produce fluorescence when subjected to PDMPO staining, the chemical shifts identified in 1H NMR analysis correlate to the presence of silica. Cyclic oligosiloxanes are neat liquids. Large, cyclic oligosiloxanes were confirmed to be present in O. littorlis, S. thurberi, and O. ficus-indica via GC-MS analysis. An O. littoralis plant lacking both crystal bodies and cyclic siloxane compounds was identified growing among the other specimens tested in this study. The essential oil composition of the plant lacking cyclic siloxane had substantially different essential oil composition from the other specimens of O. littoralis with crystal bodies. This suggests that the process of silicon transport and storage has a significant effect on the phenotype of the cactus.
5.2 Future Work
This study is the first known characterization of the volatile fraction of North
American cactus species. Future research should expand the number of species investigated. Although none of these plants presented any therapeutic application, other species may have compounds that will benefit humanity. None of the specimens studied
127 herein produced sufficient quantities of oil to support further study of potential beneficial applications.
Future analysis of plants growing in differing desert environments should be performed to help identify how plants that experience heat and water stress adapt.
Understanding how plants adapt phenotype in response to environmental conditions can provide insight into the production of heat and drought tolerant varieties of plants.
Future work should investigate whether silicon transporters in cactus are analogous to those identified in other plant families. Understanding how plants adapt to silicon in their environment is an important step in understanding the role of silicon in plants. Future research should attempt to identifiy which enzymes are involved in the production of silica bodies within the Cactaceae plant family. How the formation of silica produces a starburst shape in one species of cactus and a rod in another species should be investigated. Whether cyclic siloxane formation is a mishap of the enzymatic production of silca or the result of physiosorption should be investigated. No organism has yet to be identified that can convert inorganic silicon into bio-inorganic silicon. It is doubtful that the cacti species herein would have a mechanism to specifically convert silicic acid into cyclic siloxane. Hopefully, further research will shed light on this process. Further understanding of how the plants are able to transform silicon into silica and cyclic siloxane may lead to a better understanding of how plants cope with stress and environmental chemicals. The polymerization of silicon produces water as a by-product.
Future research should investigate whether this is a mechanism that the cacti employ to endure the arid desert conditions.
128
APPENDIX
6.1 Chromatogram of Opuntia littoralis with silica bodies.
6.2. Chromatogram of Opuntia littoralis without silica bodies.
129
6.3. Chromatogram of Opuntia ficus-indica.
6.4. Chromatogram of Opuntia prolifera growing on Santa Catalina Island, California.
130
6.5. Chromatogram of Opuntia acanthocarpo var. major growing at Arizona-Sonora Desert Museum.
6.6. Chromatogram of Opuntia acanthocarpa var. major growing in Organ Pipe Cactus National Monument.
131
6.7. Chromatogram of Opuntia phaeacanth var. discata growing wild at Arizona-Sonora Desert Museum.
6.8. Chromatogram of Opunta phaeacantha var. discata growing at Organ Pipe Cactus National Monument.
132
6.9. Chromatogram of Opuntia littoralis lab rot.
Abundance
TIC: OPTN.D\ data.ms 9500000 3.718 9000000
8500000
8000000
7500000
7000000
6500000
6000000 16.382 5500000
5000000
4500000
4000000
3500000
3000000 44.989 2500000 50.018 54.636 2000000 39.347 1500000 21.181 58.851 28.466 1000000 17.262 30.30833.23235.911 4.455 7.474 49.347 62.753 500000 33.68938.155 48.00350.193 24.24824.51525.422 31.39234.306 40.84642.58742.97644.334 55.22755.413 66.426
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 Time-->
6.10. Chromatogram of Stenocereus thurberi necrotic dynamic headspace extraction.
133
6.11. Chromatogram of necrotic Stenocereus thurberi early rot.
Abundanc e
TIC: Org_1A_5_20_B.D\ data.ms 65.727 80000
75000
70000
65000
60000
55000
50000 4.430 45000
13.214 40000 61.895
35000
30000 19.896 25000
27.982 20000 32.69236.593
15000 16.834 19.041 10000 12.18114.507
5000
0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 T ime-->
6.12. Chromatogram of late, natural rot S. thurberi.
134
6.13. Chromatogram of green S. thurberi.
A b u n d a n c e
TIC: ORGG.D\data.ms 3 0 0 0 00 3 0 . 7 1 7
2 8 0 0 0 0 0 5 0 .0 2 1
2 6 0 0 0 0 0
4 4 .9 8 3 2 4 0 0 0 0 0
5 4 .6 3 6 2 2 0 0 0 0 0
2 0 0 0 0 0 0
1 8 0 0 0 0 0
1 6 0 0 0 0 0
1 4 0 0 0 0 0
1 2 0 0 0 0 0
5 8 .8 5 1 1 0 0 0 0 0 0
8 0 0 0 0 0
6 0 0 0 0 0
4 0 0 0 0 0 6 2 .7 5 7
4 6 .0 8 1 2 0 0 0 0 0 3 9 .3 2 9 5 1 .1 4 6 6 1 .8 4 16 5 .6 6 0 4 2 .9 7 14 6 .9 05 5 0 .1 35 7 3 .2 05 8 6 .1 5 8 6 6 .4 2 5 2 7 .2 8 4 3 7 .0 2 9 4 3 .5 3 44 7 .1 2 7 5 3 .3 9 6 5 8 .9 8 9
5 . 0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0 4 5 .0 0 5 0 .0 0 5 5 .0 0 6 0 .0 0 6 5 .0 0 T im e -->
6.14 Chromatogram of lab rot S. thurberi
Abundance
TIC: ORGN.D\ data.ms 3.720 9500000 18.749 9000000
8500000
8000000
7500000
7000000
6500000
6000000 45.072
5500000 50.108 5000000 39.419 23.621 54.701 4500000 20.49020.509
4000000 58.893 3500000
3000000 30.09030.59033.41936.977
2500000 6.018 49.857 62.769 2000000 38.278 1500000 25.186 9.280 24.954 39.63042.986 50.272 55.275 1000000 33.851 48.046 55.457 44.39445.396 66.433 10.379 31.66633.13434.467 40.926 49.177 25.63725.90727.20032.18932.27532.709 42.71345.27746.13947.834 53.24453.43655.81659.940 500000 24.76826.986 41.98343.30746.68247.16347.53048.729 54.180 61.395 25.51926.90827.43130.76231.02032.52932.95834.039 41.268 47.67450.83151.18453.56856.18456.34657.47457.83458.66059.00959.17161.72061.85564.45765.66469.37669.88272.36373.969
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 Time-->
135
6.15 1H NMR spectra of Opuntia littoralis crystalline powder.
6.16 1H NMR spectra of Stenocereus thurberi crystalline powder.
136
6.17 13C NMR spectra of O. littoralis crystalline particulate.
6.1813C NMR spectra of S. thurberi crystalline particulate.
137
Table 6.19. D. melanogaster toxicity rata data. Number of survivors is shown.
Compound 0.5 1% 2% 3% 3.5 4 % 6% 8% 16% 20 30 % % % % Org Nat Rot Cactus 4 of 50 survived 24hrs, 0 in 48hrs Opt Nat Rot Cactus 5,4,0 survived 24hrs, 48 hrs 0,0,0 Opt Nat Rot 2,5,2 Extract Opt Green 10,10, 10,10 10 ,10 Phenyl ethyl 6,5,7, 9,8,9, 7,7,9 10,10,8, 8,10, 5, 0,5 alcohol 8,8,8 9,9,7 9,7,10 8 5, 6 5
2-Heptanone 8,10,1 5,8,8, 10,10 0,0,0 0,7,9, 8,8,8, 10,3, 9,10, 8,7,8, 10,7 10,3 6 Phenol 8,8,9, 9,9,8, 5,1,7, 8,9,7, 7,9,9 3,6,5, 9,10,8 6,6,4 Oleic acid 8,5,10 8,8,1 10,9, 9,10 0,0,4 ,5,9,1 0,10, 9 0,6,6, 8,9,7, 8 4,6 Butanoic acid 10,10, 9,4,1 9,10, 9,9.1 10 10, 9,6,10 0, 5 0 ,1 10, ,10,10 10,9, 0 10 ,10,9 7 10 3-Heptanone 7,8,10 10,9, 10,9, ,8,8,1 9, 9 0,9,8, 10,9, 7 9 Benzaldehyde 9,6 6,8,10 7,6,6, 10, 5,4,6 1,0,1,0,0 0 8 ,6,5,4, 6,7,6 6,7 0 8,6,10 10
138
Table 6.19 (Continued)
Pentanal 6,10, 8,9, 8,98, 8,99 4,2,6 10, 10,10 89, 10,3,1 6,8 10 0,9,9, 8 Hexadecane 10,10, 9,10, 10,10 5,6,5 5,3,0 0,0 9,9,8, 10,9, 9, 10, ,0 10,10, 10,8 10, 8 9,10 Palmitic acid 9,9,10 9,9,9 10, 10, 10, 9 10, 10, 9,9 ,10 Nonanoic acid 9,5,6, 6,10, 6,5, 7,4,2, 8,9,7, 9,7,9, 5 4,3,6, 8,9,7 6,7,9, 7,4,8 6 Hexanoic acid 6,9,9, 8,8,8, 2, 10,10,9, 8,8,9 18 15 9,9,9 8,9,8 10,8 10,10,10 Octanoic acid 9,9,10 10,7, 10,8, 5,5, 0 0,0,0 ,8,9,1 10,10 8 6 0 ,7,9 2-Nonanone 6,9,10 9,7,1 8,3,5 0 Ρ-Cresol 6,9,10 7,9,9, 10,9, 0,0,0 0,0,0 0, 0,0 10,7, 0 0, ,0 10 0 Anisole 10,10, 10,10 0,2,3. 0,0,0 0,0,0 0, 0,0 10,10, ,10 0,9, 5 0, ,0 10,10 0 Isophorone 10,9,9 9,7,9, 10,9, 8,92 4,3,4 ,9,9,1 9,8,9 7 0 Si 30 30 Control 10,10, 10,10 10, 10,10 10, 9, 10, 10 10, 10 10, 10,9,1 9 10, ,10 10, 10,10 , 9,9 0,10,1 9 9 9, 0,10 10 Green Opt Cactus 10,10, 10 Green Org Cactus 10,10, 10,10 10 ,10
139
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