A Chemical Foundation for Native American Use of Cercis Canadensis and Zanthoxylum Clava-Herculis

A CHEMICAL FOUNDATION FOR NATIVE AMERICAN USE OF CERCIS CANADENSIS AND ZANTHOXYLUM CLAVA-HERCULIS

by KELLY MARIE STEINBERG

A MASTERS THESIS

Submitted in partial fulfillment of the requirements

for the degree of Masters of Science

The Department of Chemistry to The School of Graduate Studies of

The University of Alabama in Huntsville

HUNTSVILLE, ALABAMA

2017

In presenting this thesis in partial fulfillment of the requirements for a master's degree from The University of Alabama in Huntsville, I agree that the Library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by my advisor or, in his 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 thesis.

ii THESIS APPROVAL FORM

Submitted by Kelly Marie Steinberg in partial fulfillment of the requirements for the degree of Master of Science in Chemistry and accepted on behalf of the Faculty of the School of Graduate Studies by the thesis 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 thesis. We further certify that we have reviewed the thesis manuscript and approve it in partial fulfillment of the requirements for the degree of Master of Science in Chemistry.

iii ABSTRACT The School of Graduate Studies The University of Alabama in Huntsville

Degree _____Master of Science in Chemistry College/Dept. ______Science/Chemistry______.

Name of Candidate______Kelly Marie Steinberg ______. Title ______A Chemical Foundation for Native American Use of Cercis canadensis and______Zanthoxylum clava-herculis______.

This thesis project studied the bark of North American plants, Cercis canadensis and Zanthoxylum clava-herculis, plants used medicinally by the Cherokee Indians. The C. canadensis bark was analyzed through Soxhlet extraction, column chromatography, and thin layer chromatography to isolate its nonvolatile components. Collection and analysis of NMR experiments- proton, carbon, HSQC, HMBC, and COSY, led to identification of the compound lupeol. The C. canadensis and Z. clava-herculis bark essential oil was collected with Likens- Nickerson hydrodistillation and analyzed through GC-MS and chiral GC-MS. Major components of the C. canadensis bark oil include 1-hexanol, hexanoic acid, (2E)-hexenoic acid, oleic acid amide, and 1-docosanal. Major components of the Z. clava-herculis bark oil were sabinene, limonene, γ- terpinene, and terpinen-4-ol. The compounds identified have various reported bioactivities in the literature suggesting a possible reason for their medicinal use by the Cherokee.

Keywords: Cercis canadensis, Zanthoxylum clava-herculis, Soxhlet extraction, column chromatography, TLC, Nuclear Magnetic Resonance Spectroscopy, Likens-Nickerson, Gas Chromatography-Mass Spectrometry

Abstract Approval:

iv ACKNOWLEDGMENTS

This thesis project would not be possible without the help and guidance of many people. First, I would like to thank my adviser, Dr. William Setzer, for the suggestion of this research topic and guidance throughout the work. Second, I would like to thank my committee members for their suggestions and corrections. I am forever grateful to my family and friends, who encouraged me to persevere in this work and prayed for me throughout. Finally, I am thankful to my Lord and Savior, Jesus Christ for giving me the ability and strength for this project.

2 Corinthians 4:7 “But we have this treasure in jars of clay to show that the all-surpassing power is of God and not from us.”

v TABLE OF CONTENTS

PAGE

vii LIST OF FIGURES

FIGURE PAGE

viii LIST OF TABLES

TABLES PAGE

Table 1: Medicinally Useful Plants to the Cherokee Indians:………………………………………..……15

Table 2: NMR peak shifts from carbon δ 13C and proton δ 1H spectras:………………..…..………...37

Table 3: Essential oil of Cercis canadensis Bark Oil:……………………………………………..….….……..43

Table 4: Essential oil of Zanthoxylum clava-herculis Bark Oil:………………………………..….….…...47

CHAPTER ONE

INTRODUCTION

Nature holds undiscovered chemistry. Plants with long-acclaimed

medicinal uses by local communities are a treasure box of cures for the world’s

destructive diseases, just waiting to be discovered. Learning the phytochemistry of

these plants can not only confirm a scientific basis for their historical use, but also

unlock new successful treatments for today’s uncured diseases. This study looks

into the phytochemistry of two North American plants used to treat local illnesses

during the time of the Cherokee and Houma Indians inhabiting the North Alabama area where this study took place. The plants studied in this project include Cercis canadensis and Zanthoxylum clava-herculis. Each of these plants are North American trees located in the southeastern United States. The C. canadensis is commonly called redbud and was used by the Cherokee Indians for treatment of various diseases with symptoms of cough and fever1–3. The Z. clava-herculis is commonly called Southern prickly ash, along with some other common names, and was used as

a pain reliever for swelling and toothaches by the Houma and Cherokee Indian

tribes3–5. The bark of the C. canadensis and Z. clava-herculis provide an exciting topic of study from their known ethnobotany, which revealed their previous medicinal use. For this reason, these trees were selected for phytochemical study. The pharmacological interest in these plants led to the phytochemical experiment recorded in this project.

1.1 Tree Collection Area: Huntsville, AL

Both of these plants were collected in Huntsville, AL. These plants can be found throughout the city in residential, industrial, and wooded areas. This study was done through years of uncommonly long summers with temperatures during early November only reaching down into the low to mid 30soC. The C. canadensis typically blooms small purple flowers in the spring and holds its leaves through the summer into early fall around September. With longer warm days in the duration of this study, C. canadensis and Z. clava-herculis held onto their leaves longer than usual.

Although the C. canadensis bark was collected earlier in the fall for extraction, the C. canadensis bark collected for hydrodistillation was collected during November of

2016 while the tree still had its leaves. The Z. clava-herculis was also collected in

November 2016 during uncommonly warm temperatures. The date and conditions of collection of a given plant can determine its chemical composition.

A plant’s chemical composition can vary quantitatively depending on many

factors-from the environmental conditions to their specific collection site

properties6. C. canadensis and Z. clava-herculis bark compounds were identified in this study from only one collection of each type of compounds identified- nonvolatile compounds from C. canadensis bark, volatile compounds from C. canadensis bark, and volatile compounds from Z. clava-herculis bark. To further determine if these plants would be a plentiful source of the compounds identified, collections would have to be taken from multiple sites at different times and analyzed. This work contains an initial qualitative identification of the chemical constituents of the two medicinal trees - C. canadensis and Z. clava-herculis.

1.2 Cercis canadensis

The C. canadensis, commonly called eastern redbud, is of the family Fabaceae, the legume family1,7. Another common name of this tree is the Judas-tree7. C.

canadensis has been used for generations for various remedies. It is widely

considered a great addition to landscaping projects because of its beautiful, purple

flowers in the spring, and easily distinguishable heart-shaped leaves that come in

late spring to mid-fall.

1.2.1 Cercis canadensis Anatomy

C. canadensis is a small tree, similar to a shrub, growing about 14 m in

height and 80 cm in diameter7. The twigs usually zigzag and have a smooth,

brown bark, while the bark of the trunk has narrow, scaly ridges5. The

flowers cluster anywhere on the stem, Figure 1, except on the tips of the

twigs5. Figure 1 also shows the fruit pods, which are usually 4-10 cm in

length and 8-18 mm in width7. The leaves, also shown in Figure 1, are dark

green and glabrous on top, and pale green and pubescent on the underside5.

Approximately 5 to 7 veins arise from one point at the base of the blade of

the leaf7.

Figure 1: Cercis canadensis with its purple buds in spring and heart-shaped leaves in autumn in Huntsville, AL. The picture on the right shows the fruit of C. canadensis.

1.2.2 Cercis canadensis Variations

There are two variations of redbud, the Mexican redbud and the

eastern redbud. One study was done comparing how these two variations

responded to drought. The main difference found between these redbud

variations was not in their tolerance of high temperatures, but was in

photosynthesis, possibly because the Mexican redbud has thicker leaves than

the eastern redbud. They grow in different climates: the Mexican redbud in

xeric, dry environments, and the eastern redbud in mesic, moderate

environments. The Mexican redbud can be found in southwest Texas, and

further south into Mexico City8. The bark of the eastern redbud was studied

in this project: this variation of redbud is found in the southeastern region of

the United States, including Huntsville, AL where this study was conducted.

1.2.3 Cercis canadensis Appearance and Nonmedicinal Uses

Two cultivars of eastern redbud are Ace of Hearts and Little Woody;

they both maintain their distinctive heart-shaped leaves preceded by purple

buds in the spring with clusters of 4-8 and 2-7 buds, respectively9. C.

canadensis holds much more than beauty for its beholders; its blossoms were

at one time used by the Cherokee Indians for food, reportedly eaten by their

children, and the tree was used medicinally3. It produces numerous

seedlings, and it spreads easily7. Perhaps further study of this lovely tree and

its chemical constituents will provide a new use for it today beyond being a

striking garden centerpiece.

1.2.4 Medicinal Uses of Cercis canadensis

C. canadensis bark was reportedly used by the Cherokee Indians to

make tea as a remedy for whooping cough2. Whooping cough is caused by the

bacteria Bordetella pertussis that exhibits a pathogenesis discussed below10.

Other than a pulmonary aid for whooping cough for the Cherokee tribe, C.

canadensis was reportedly used for other symptoms by other tribes. In

addition to whooping, other illnesses found their remedy within the bark of

C. canadensis. The Alabama, Delaware, and Delaware-Oklahoma tribes used C.

canadensis for various ailments such as congestion, fever, and vomiting3.

1.2.4.1 Mechanism of Pathogenesis of Pertussis

Pertussis is the alternative name of whooping cough, a

contagious respiratory disease, which is treated today by

vaccination10. The bacteria, B. pertussis, produces an islets-activating

protein that is responsible for some of the symptoms of pertussis

and is generally recognized as the pertussis toxin11. Pertussis toxin

has been shown to bind to the surface of mammalian cells,

specifically Chinese hamster ovary cells that are lacking sialic acid

and galactose residues on their macromolecules12. These cells show

terminal N-acetylglucosamine residues present on their surface

macromolecules, indicating that for pertussis toxin to bind

optimally, a sialyllactosamine sequence must be present on the

surface of the cell. The study also showed that the pertussis toxin

has 2 glycoprotein-binding sites that are different in their

specificity12. The pertussis toxin can be transported into the cell by

receptor-mediated endocytosis13. Once inside the cell, pertussis

toxin is transported to the endoplasmic reticulum, where its

structure is altered by dissociation and part of it moves to the

cytosol14.

According to the CDC, B. pertussis attaches itself to the cilia of

respiratory epithelial cells and then releases the pertussis toxin. The

pertussis toxins are known to paralyze cilia, causing the respiratory

tract to swell. When B. pertussis invades a host, it not only releases

the pertussis toxin, but also the antigens: filamentous

hemagglutinin, agglutinogens, and adenylate cyclase, which together

cause lymphocytosis. Lymphocytosis is a host response to the

invasion of B. pertusis that is often used to diagnose the disease. The

antigens of B. pertussis also hinder the host’s chemotaxis10.

1.2.5 Chemical Composition of Cercis canadensis

The phytochemical studies on redbud have focused on mature, green

seeds and chloroplasts of the tree. After being analyzed by aqueous and

alkaline extraction, the C. canadensis seed endosperm revealed the

monosaccharide composition of 10.4 mannose to 0.9 galactose, while the

seed hull contained the polysaccharides composed of 4.5 mannose to 0.9

galactose15. These monosaccharides are shown in Figure 2 below.

Galactoglucomannans, -D-galactopyranosyl, 4-O- -D-mannopyranosyl, 4,6-

di-O- -D-mannopyranosylα , and 4-O- -D-glucopyranosylβ were found in the

seed βthrough 13C NMR, nuclear magneticβ resonance spectroscopy15.

HO HO

H O H OH O HO

OH OH OH H OH HO H H

a) H H b) H OH

Figure 2: Monosaccharides of C. canadensis Seed Endosperm a) Mannose b) Galactose

An additional study of the seed water-soluble polysaccharides was

performed on the C. canadensis. This study found that polysaccharides made

up 0.3% of the composition of the C. canadensis seeds. The water-soluble

polysaccharides found were composed of rhamnose, xylose, arabinose,

mannose, and galactose; some of the polysaccharides were also found in the

study by Mestechkina16. The isolated chloroplasts of the C. canadensis were

examined in one study focused on flavonoids. Some isolated flavonoids from

the chloroplasts are quercetin-3-glycoside, quercetin-3-diglycoside, and

quercetin-3,7-diglycoside17. These previous phytochemical studies have

focused on the seeds, leaves, and chloroplasts of C. canadensis, while this

study focuses on the bark’s volatile and nonvolatile chemical constituents.

1.2.6 Location of Cercis canadensis

The C. canadensis grows in both moist and dry habitats. It can be

found in the southeastern United States at elevations lower than 670 m; it

can be found in west Texas and New Mexico. Another species grows in China,

Europe, and West Asia7. C. canadensis can also be found in the undergrowth

of high forests in mesophile-containing forests above the Tennessee River

and in the prairie regions and prairie region openings of Alabama18. C.

canadensis grows along the slopes fronting streams in the mountainous

forest regions of Alabama18.

1.3 Zanthoxylum clava-herculis

Zanthoxylum clava-herculis is from the family Rutaceae, the citrus family1,5. It

has been called Hercules’s Club, southern prickly ash, or tickle-tongue3–5.

Additionally, Hercules’s club was a common name used to refer to a plant contained

in Table 1, Aralia spinosa1. Z. clava-herculis has been found to be food for the larvae of the giant swallowtail butterfly, Papilio cresphontes, along with its other

interesting uses19.

1.3.1 Zanthoxylum clava-herculis Anatomy

The leaves of Z. clava-herculis have a leathery appearance, with

terminal clusters of flowers and fruits7. Z. clava-herculis grows to an average

height of 12 m and a width of 70 cm. There are prickles on the leaf axis, on

the tops of the trunk knobs, and on the twigs7. Both the leaves and twigs are

aromatic when crushed, and there is oil throughout the plant, which is

studied in this project7.

Young Z. clava-herculis twigs differ in appearance to twigs a year old

or more; young twigs are glabrous and pustular-punctate, while year-old

twigs have brown bark with irregular divided longitudinal streaks5. Leaves of

the Z. clava-herculis are around 2.5 to 7 cm in length; the tops are dark green

and lustrous, while underneath is paler and dull, glabrous, with yellowish

glands in the notches5. The flowers have 1-3 pistils, with 4-5 greenish-yellow petals5,7.

Figure 3: Zanthoxylum clava-herculis leaves, trunk, and fruit1

1.3.2 Medicinal uses of Zanthoxylum clava-herculis

Z. clava-herculis root was reportedly used by the Houma Indians as a

remedy for swelling. It was prepared by grating and mixing it with whisky

and then applying it topically. The root and bark were also grated and used

for toothaches3. The Xanthoxylum plants, another name for Zanthoxylum,

were commonly called “toothache trees” due to the compound hydroxyl- -

sanshool, which excites sensory neurons20. While the Z. clava-herculis hasα

reported use with the Houma Indians, it has no reported use by the Cherokee

Indians. A very similar tree, however, the Z. americanum, was reportedly

used by the Cherokee to make a tea for washing joints, as a treatment for

swelling and arthritis2,3. The Z. americanum, commonly called prickly ash, is

easily confused with Z. clava-herculis, since they have similar anatomy and

their almost identical common names1.

1.3.3 Chemical Composition of Zanthoxylum clava-herculis

The Z. clava-herculis leaf essential oil has been phytochemically

studied and found to have many menthane monoterpenoids, including the

major components limonene and 1,8-cineole19. The leaf essential oil of Z.

clava-herculis has also been collected at different times to see the effects of

seasonal variables in its chemical composition21. The major chemical

constituents present at almost all times of collection remained the same;

however, they varied in percent composition with limonene making up 44 to

73% and 1,8-cineole making up 16-43% of the leaf essential oil depending on the collection month21. The minor chemical constituents present in all of the leaf essential oil collections were -thujene, linaloo -terpinene, cis- sabinene hydrate acetate, -terpineolα 21. l, γ

Some early phytochemicaland α studies indicated that in the petroleum ether extract of the Z. clava-herculis bark, insecticidal compounds were present- including asarinin (Figure 4) and a compound similar to pyrethrum4.

O H O O O O H O

Figure 4: Asarinin - a Toxic Compound to Houseflies

Further study of the insecticidal activity of the Z. clava-herculis bark proposed that the presence of the compound (2E,8E)-N-isobutyl-2,8- dodecadienamide (Figure 5), commonly called herculin, is partially responsible for the bark’s toxicity to houseflies (Musca domestica)22. After an attempt to re-isolate herculin, another study found that it had been misidentified, and was actually the stereoisomers, (1E,7E)-N-isobutylundeca-

1,7-diene-1-carboxyamide and (1Z,7E)-N-isobutylundeca-1,7-diene-1- carboxyamide23. This compound was given the name neoherculin23.

CH3

H3C NH

CH3

Figure 5: Herculin - (2E,8E)-N-isobutyl-2,8-dodecadienamide – initially thought to be present in Z. clava-herculis bark

The extraction of Z. clava-herculis bark has also shown the presence

of the active compound benzo[c]phenanthridine alkaloid chelerythrine24.

This compound showed significant activity against some strains of

Staphylococcus aureus as compared to the antibiotics tetracycline,

erythromycin, and norfloxacin24. The compound mentioned in the previous

section, hydroxyl- -sanshool, has also been located in the Xanthoxylum

plants and its functionalityα as an anesthetic determined20. Although many

bioactive compounds have been previously identified in Z. clava-herculis,

there is no study until now on the bark essential oil and identification of

volatile compounds of Z. clava-herculis bark.

1.3.4 Location of Zanthoxylum clava-herculis

The Z. clava-herculis can be found in dunes and maritime woods in the

southeastern United States extending from west Texas and Oklahoma to west

North Carolina and Virginia7. It also grows in shell middens, along fences, and

in hedgerows5. The Z. clava-herculis thrives as a shrub growing underneath

much taller trees in soil from hammock lands. This undergrowth region

usually contains black soil composed of decayed vegetation along river

swamps18. This soil supports a varied collection of trees and shrubs, along

with rain worms, snails, centipedes, bacteria, and fungi18.

1.4 Metabolism of Fatty Acids in Natural Products

Small alcohols, aldehydes, and carboxylic acids are common in the essential

oils of many plants. In the olive tree fruit, Olea europeae, volatile compounds with

six carbons are secondary products of the plant’s intracellular biogenetic

pathways25. The first step in the metabolism of these compounds was the hydration

of the olive’s triacylglycerols and phospholipids with acylhydrolase forming linoleic

acid. These fatty acids are then converted with lipoxygenase to hydroperoxides,

which are cleaved by lyase to form the six-carbon aldehyde. This aldehyde, after

incubation, can convert to alcohols and acetate esters25. This metabolism from

linoleic acid has been seen also in banana tissues producing six-carbon aldehydes26.

1.5 Phytochemical Compositions of Cherokee Medicinal Plants

The following table contains a record of 16 medicinal plants used by the

Cherokee Indians for various ailments. Studying the ethnobotany of these plants

allows for the specified phytochemical search for cures of diseases today beyond

this study. This table includes essential oils, volatile compounds, and nonvolatile

compounds identified from various parts of each of the following plants.

Table 1: Medicinally Useful Plants to the Cherokee Indians:

Common family names from Tropicos1 except when indicated otherwise; Part of plant used2

CHAPTER TWO

EXPERIMENTAL

2.1 Collection

Limbs of the plants Cercis canadensis and Zanthoxylum clava-herculis were

collected in North Alabama. Branches of C. canadensis were collected from the

University of Alabama in Huntsville’s campus (34o 43.32' N, 86o 38.30' W, 185 m elevation) on August 23 and 25, 2015 and from a suburban South Huntsville house

November 5, 2016 (34o38'46"N, 86o33'27"W, 191 m elevation). Branches of Z. clava-

herculis were collected from the same suburban South Huntsville house on

November 13, 2016. The bark was stripped from the limbs and finely chopped, as

shown in Figure 6. The Z. clava-herculis branches required the removal of the thorns before the bark was stripped and chopped.

Figure 6: Branch of Cercis canadensis - being stripped of its bark and prepared for chopping.

2.2 Extraction

A little over 1.5 kg of chopped C. canadensis bark was air dried before extraction. A Soxhlet extraction, as shown in Figure 7, was performed with the chopped bark using dichloromethane. Each extraction was run for 4 to 6 h. The crude extract was evaporated in order to obtain the crude extract mass without solvent and in preparation for chromatographic separation.

Figure 7: The Soxhlet Extraction of Cercis canadensis

2.3 Chromatographic Separation

The crude bark extract of C. canadensis was analyzed through Thin Layer

Chromatography (TLC) developed in a chloroform and methanol chamber on a silica-coated, plastic-backed plate. This was repeated with a hexane and ethyl acetate developing chamber to determine the best solvent system to use in separation. The crude extract, 25.80 g, from C. canadensis bark was separated initially through column chromatography with silica and a solvent system of hexane and ethyl acetate. The silica gel was 40- m particle size and 60 porosity. The silica gel was used as the stationary phase63 ,μ and the column used wasA� glass, 90 cm in length and 5 cm in diameter. The column was packed with hexane and then the concentration of ethyl acetate was increased: from a 9:1 hexane to ethyl acetate

ratio, then to a ratio of 8:2, 1:1, and finally 100% ethyl acetate throughout fraction collection. Figure 8 shows the C. canadensis column. Fifty-five ~200-mL samples of

C. canadensis were collected and evaporated. The masses of the evaporated samples from the column were found. They were then individually dissolved in ethyl acetate and some hexane, and were analyzed through TLC to combine column samples containing the same compounds. The samples were combined based on the presence of the same compound in multiple samples. The column samples were combined into 13 major fractions shown in the flow chart, Figure 9 below. Some major fractions were being collected at the end of one solvent ratio and the beginning of another. The flow chart shows the approximate solvent ratio during which each major fraction eluted from the column.

Figure 8: Column Chromatography of C. canadensis

25.80g crude extract

9:1 Hexane/ Ethyl 1:1 Hexane/ Ethyl Ethyl 8:2 Hexane/ Ethyl Acetate Acetate Acetate Acetate

F 1-4 F 5-6 F 7-13 F 14-15 F 16-17 F 18-22 F 23-26 F 27-39 F 40-45 F 46-47 F48-55 F 56 F 57-59

MF 1 MF2 MF3 MF4 MF5 MF6 MF7 MF8 MF9 MF10 MF11 MF12 MF13

Figure 9: C. canadensis column fractions with approximate solvent ratios for each fraction and TLC combinations. Major fractions (MF) were combined for recrystallization.

2.4 Isolation of Compounds

A recrystallization process using simple diffusion was used for each major fraction. The fractions were placed in clean glass scintillation vials and dissolved in warm ethyl acetate. These were placed into a larger vial and covered with a Teflon lid. Pentane was put into the larger outer vial with approximately half the depth of the solution in the inner scintillation vial. The simple diffusion technique is shown in Figure 10. After the lid was placed on the larger vial, crystals formed in some of the fractions over a time span of 24 to 48 hours. The supernatant was pipetted off and transferred to a new vial for repetition of the simple diffusion technique on the supernatant. All the recrystallized compounds were dried by evaporation and then checked for successful isolation by testing each sample with TLC. Successful isolation was confirmed when a single spot appeared on the developed TLC plate.

Figure 10: Simple Diffusion Technique - vial in vial recrystallization, where small vial contents are green and pentane in the outer chamber is clear.

2.5 Identification of Pure Compounds

The fractions showing a single spot on the TLC plate were then prepped for

Nuclear Magnetic Resonance (NMR) spectroscopy. Any remaining solvent was evaporated off the crystals. The pure compounds were dissolved in deuterated chloroform, CDCl3, and placed in NMR tubes. The experiments 1H, 13C, gradient selected correlation spectroscopy (g-COSY), gradient selected heteronuclear multiple-bond correlation spectroscopy with adiabatic pulses (gHMBCAD), and heteronuclear single-quantum correlation spectroscopy with adiabatic pulses

(HSQCAD) were run on the Varian 500, shown below in Figure 11. The 1- dimensional spectra 1H, 13C, and the 2-dimensional spectra g-COSY, gHMBCAD, and

HSQCAD were used for structure identification. The NMR spectra were analyzed using MestReNova software to determine the structural correlations of the peaks, integration, and through bond correlations- as discussed in the results section. The peaks were compared to previously known compounds published in the literature.

Figure 11: Varian 500 in the NMR Laboratory

2.6 Hydrodistillation

The C. canadensis chopped bark, 87.78 g, was placed in a flask for Likens-

Nickerson hydrodistillation, as shown in Figure 12. The continuous extraction of

essential oils with dichloromethane was done for 4 h. The yield of C. canadensis bark

oil was 1.6709 g collected and stored in the laboratory refrigerator at -20oC until

further analysis. The yield of essential oil from C. canadensis was 1.90% mass percent yield. The Z. clava-herculis chopped bark, 146.01 g, was placed into the flask

used for hydrodistillation. A 4 h extraction was performed for this bark. The yield of

Z. clava-herculis oil was 4.24 g, a 2.90% mass percent yield of essential oil.

Figure 12: Likens-Nickerson Apparatus

2.7 Gas Chromatography-Mass Spectral (GC-MS) Analysis

The GC-MS was performed by collaborating researcher Prabodh Satyal. The procedure followed is the same as outlined in a previous publication52. The essential

oils of C. canadensis and Z. clava-herculis were analyzed by GC-MS with a Shimadzu

GCMS-QP2010 Ultra. This instrument was operated in the electron impact (EI) mode set at electron energy 70eV with a scan range of 40-400 amu, a scan rate of 3.0 scans per second, and with GC-MS solution software. A ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of

was used as the GC column. Helium was used as the carrier gas, and the

pressure0.25 μm was set at 80 psi with a flow rate of 1.37 mL/min on the column head. The

temperature of the injector was set at 250°C and the temperature of the ion source

was set at 200°C. The temperature of the GC oven was programmed to be 50°C

initially and was programmed to increase at a rate of 2°C/ min to a final temperature of 260°C. The samples were prepared with dichloromethane, CH2Cl2, in

a 5% w/v solution. Then, 0.1 µL of the solutions were injected into the instrument

with the splitting mode with a split ratio of 30:1. The components of each essential

oil sample were identified on the retention indices and mass spectral fragmentation

patterns compared to reference literature and an in-house library53. The retention

indices are determined by reference to a homologous series of n-alkanes.

2.8 Chiral Gas Chromatography- Mass Spectrometry (GC-MS)

The essential oils from both C. canadensis and Z. clava-herculis were also chirally analyzed with a Shimadzu GCMS-QP2010S. This experimentation was also done by Prabodh Satyal, as outlined in a previous publication52. The instrument was

operated in the EI mode with electron energy of 70 eV, a scan range of 40–400 amu,

and a scan rate of 3.0 scans/s. The capillary column used was a Restek B-Dex 325 with film dimensions of 30 m by 0.25 mm ID by 0.25 . The temperature of the oven was programmed to start at 50°C and to rise at μma rate of 1.5°C/min to a final temperature of 120°C. Then, the oven was raised to 200°C at a faster rate of 2°C/min and maintained for 5 min. The carrier gas, helium, was set at a constant flow rate of

1.8 mL/min. Solutions of 3% w/v were prepared with the essential oils in the solvent CH2Cl2. Then, 0.1 L of sample solution was injected into the instrument in split mode with the split ratioμ of 1:45.

CHAPTER THREE

RESULTS

3.1 Compounds Identified from Cercis canadensis

The following compound was isolated from Cercis canadensis extraction. The nonvolatile compound was identified with collected NMR spectra shown below.

Volatile compounds were identified using GC-MS spectra shown whose data is also shown below.

3.1.1 Lupeol

The first compound identified was from the combined column

samples 16-17, major fraction 5 that eluted from the column at the solvent

ratio of 8:2 hexane to ethyl acetate. After one recrystallization by simple

diffusion, NMR analysis, and comparison to literature values54, the compound

was found to be lupeol, as shown in Figure 13. Lupeol has a chemical formula

of C30H50O with a molar mass of 426 g/mol. The yield of lupeol was 0.0183 g,

a percent yield of 0.007% from the original extract. Of the samples collected

from the column, the total mass collected in the 58 fractions was 18.95 g. The

percent yield of lupeol from the total mass of sample eluted from the column

was 0.097%.

CH2

H3C H H

H H3C CH3

CH3 CH3 HO H CH H3C 3

Figure 13: Lupeol Structure

1H NMR (500 MHz, CDCl3)

(dd, 1H), 2.37 (td, 1H), 1.92 (m, 1H),δ 4.69 1.68 (d, (s, J= 3H), 2.49, 1.66 1H), (s, 4.56 1H), (dd, 1.65 1H), (s, 1H), 3.20

1.60 (m, 1H), 1.57(s, 1H), 1.52 (m, 1H), 1.47 (dd, 1H), 1.42 (m, 1H), 1.39 (m,

1H), 1.38 (m, 1H), 1.37 (s, 1H), 1.36 (s, 1H), 1.33 (s, 1H), 1.26 (s, 1H), 1.18 (s,

1H), 1.03 (s, 3H), 1.02 (d, 1H), 0.99 (d, 1H), 0.97 (s, 3H), 0.94 (s, 3H), 0.90 (d,

1H), 0.83 (s, 3H), 0.79 (s, 3H), 0.76 (s, 3H), 0.68 (s, 1H).

Two distinct protons of lupeol are seen in the peaks at 4.56 and 4.69

ppm, which are connected to carbon 29. These protons are olefinic protons

because they are attached to a double-bonded carbon. There are 10 other

carbons with 2 protons, but they are methylene groups (-CH2-). These are

seen at the following shifts, 0.90, 1.65, 1.60, 1.02, 1.52, 1.39, 1.39, 1.42, 1.65,

0.99, 1.57, 1.37, 1.47, 1.92, 1.33, 1.18, and 1.38 ppm. There are 6 methyl groups (-CH3), seen in the integration of the proton spectra and the color of

the signals indicated in the HSQC spectra. The methyl protons are given by

the signals 1.68, 1.03, 0.97, 0.94, 0.83, 0.79, and 0.76 ppm. There are 6

tertiary protons: 3.20, 0.68, 1.26, 1.66, 1.36 and 2.37 ppm. The peak at 7.26

ppm is from the solvent CDCl3.

13C NMR (126 MHz, CDCl3)

47.99, 42.93, 42.77, 40.00, 40.83,δ 150.96, 38.83, 109.30, 38.69, 78.98, 38.01, 55.28, 37.08, 50.43, 35.57, 48.30, 34.26,

29.86, 27.95, 27.41, 27.36, 25.10, 20.90, 19.28, 18.30, 17.96, 16.09, 15.95,

15.34, 14.53

Lupeol has a total of 30 carbons; these were seen in the observation of

HSQC and HMBC spectra. The carbon skeleton matched with the literature values of lupeol54. The carbons with attached functional groups are carbon 3,

at shift 78.98 ppm that shows a carbon bonded to a hydroxyl group (C-OH).

The carbons at 150.96 and 109.30 ppm are alkenic (-C=C-) and have a double

bond connected to the five-membered ring of the lupeol ring structure. The

solvent CDCl3 has a peak at 77.16 ppm.

There is a large proton peak at 1.26 that shows correlation to a

secondary carbon with a shift of 29.61 ppm and a primary carbon with a shift

of 50.43 ppm in the HSQC analysis. According to the HMBC correlations, the

CH2 chain is not attached to lupeol; it is a secondary compound in the

isolated crystal from major fraction 5. The secondary compound is a wax that

will require further research for identification. The contaminant compound is

seen with a peak at 0.88 and 14.07 ppm for its terminal methyl group, while

its chain methylenes are seen at 1.26, 29.65 ppm.

Table 1: 13 1H spectra seen in HSQC and HMBC correlations NMR peak shifts from carbon δ C and proton δ

13 1 13 1 Position C H C H From From Carbon δ Proton δ Carbonliterature δ 54 Protonliterature δ 54 1 38.69 0.90, 1.65 38.72 0.90, 1.67 2 27.41 1.60, (1.02) 27.43 1.60, 1.56 3 78.98 3.20 79.02 3.19 4 38.83 ------38.87 ------5 55.28 0.68 55.31 0.68 6 18.30 1.52, 1.39 18.33 1.51, 1.39 7 34.26 1.39 34.29 1.39 8 40.83 ------40.84 ------9 50.43 1.26 50.35 1.27 10 37.08 ------37.18 ------11 20.90 1.42 20.94 1.41, 1.23 12 25.10 1.65 25.16 1.07, 1.67 13 38.01 1.66 38.07 1.66 14 42.77 ------42.85 ------15 27.36 0.99, 1.57 27.46 1.00, 1.68 16 35.57 1.37, 1.47 35.60 1.37, 1.47

17 42.93 ------43.01 ------18 48.30 1.36 48.32 1.36 19 47.99 2.37 48.00 2.38 20 150.96 ------150.98 ------21 29.86 1.92, 1.33 29.86 1.32, 1.92 22 40.00 1.18, 1.38 40.22 1.19, 1.38 23 27.95 0.97 28.00 0.968 24 15.34 0.76 15.38 0.761 25 16.09 0.83 16.13 0.830 26 15.95 1.03 15.99 1.030 27 14.53 0.94 14.56 0.945 28 17.96 0.79 18.02 0.788 29 109.30 4.56, 4.69 109.32 4.56, 4.69 30 19.28 1.68 19.32 1.68 OH 1.26 1.26

6 methyl groups

Figure 14: Proton spectra of lupeol. The purple peaks indicate the 6 methyl groups of lupeol, the red peak indicates the alcohol group, the green indicates a methyl group attached to a olefinic group, and the blue peaks indicate the olefinic protons.

The next technique used for identification is a two-dimensional

heteronuclear NMR technique that produces a series of cross peaks from CH, CH2, and CH3 groups present in the compound, placing the 13C spectrum on the left axis

and the 1H spectrum on the top axis55. This spectroscopy technique is commonly referred to as HSQC, although specifically in this project HSQCAD was run which includes adiabatic pulses. During an HSQC experiment, signal intensity differs depending on how many hydrogen atoms are attached to each carbon while suppressing the homonuclear 1H-1H signals56. HSQC just shows signals for carbon atoms directly attached to hydrogen atoms57. Using the Varian 500 with

MestReNova software, the HSQC spectrum displays red, blue, and orange signals

indicating CH3, CH2, and CH groups respectively. The spectrum below was used to find all non-quaternary carbons within the lupeol structure.

Figure 15: HSQC of Lupeol

The following spectroscopy technique used in the identification of lupeol is

HMBC. The specific technique used in this project was gHMBCAD. HMBC is another

two-dimensional technique that gives signals of carbon to hydrogen correlations. In

this spectra the 1H spectrum is on the top axis, and the 13C spectrum is on the left

hand axis, like in HSQC. HMBC differs from HSQC in that it gives signals of long range coupling between carbon and hydrogens that are separated by two or three bonds57.

The HMBC spectrum was used in this project to identify quaternary carbons, which

are not seen in the signal from the HSQC spectrum57.

Figure 16: HMBC of Lupeol

The final two-dimensional NMR technique used to confirm the correct identification of lupeol was COSY. This technique looks at correlations of hydrogen- to-hydrogen through the compound. Both axes show the proton spectrum, and the peaks used for analysis are the cross-peaks given when proton nuclei exchange magnetization during the evolution time or are coupled57. The cross peaks are symmetrical on either side of the diagonal57. The coupling shown is long range coupling, giving signals for protons that are separated by two or three bond lengths57. This helps confirm the structure by allowing for the observation of protons that are near each other in the compound.

Figure 17: COSY of Lupeol

3.1.2 Cercis canadensis Bark Essential Oil

The bark collected for essential oil extraction was collected on 5

November, 2016. From the original mass of 87.78 g of chopped bark,

1.6709 g of essential oil was collected. The oil collected was clear and

colorless, as shown in Figure 18 below.

Figure 18: Cercis canadensis Oil Analyzed by GC-MS

There were 57 compounds identified in the essential oil from the bark

of Cercis canadensis. The oil was composed of 76% fatty acid derivatives,

10.2% n-alkanes, and 5.5% aromatic compounds. The concentration of

monoterpenoids was 4.1%, which was low but enough to determine the

enantiomeric distribution using chiral gas chromatography-mass

spectrometry. These results are indicated below in Table 3. The percent abundance of each compound identified is given below in Table 3.

Table 3: Essential oil of Cercis canadensis Bark Oil

RIa Compound %

799 hexanal 0.90 832 2-methyl-butanoic acid 1.00 844 (3Z)-hexenol 0.33 849 (3E)-hexenol 2.20 859 (2Z)-hexenol 0.67 862 1-hexanol 23.33 885 4-hepten-2-ol 0.79 900 2-heptanol 1.71 931 -pinene 0.18b 967 1-heptanol 0.56 975 hexanoicα acid 18.15 977 1-octen-3-ol 1.47 1003 octanal 0.42 1008 (2E)-hexenoic acid 3.44 1028 limonene 2.01c 1032 benzyl alcohol 1.26 1042 benzene acetaldehyde 0.98 1069 1-octanol 1.19 1083 ο-guaiacol 0.34 1092 unidentifiedd 2.11 1099 linalool 0.78e 1104 nonanal 1.79 1111 phenylethyl alcohol 0.64 1159 (2E)-nonenal 0.58 1164 octanoic acid 1.07 1194 -terpineol 0.72f 1205 decanal 1.23 1230 2-coumaranoneα 0.54 1248 chavicol + geraniol 0.82 1349 eugenol 0.69 1397 methyleugenol 0.61

RIa Compound %

1418 -caryophyllene 0.71 1446 geranyl acetone 0.45 1508 dicyclohexylβ ketone 0.49 1580 caryophyllene oxide 1.09 1600 hexadecane 0.62 1607 1,10-di-epi-cubenol 0.73 1654 -cadinol 0.85 1700 heptadecane 0.82 1793 1-αoctadecene 0.49 1800 octadecane 0.63 1894 1-nonadecene 0.71 1900 nonadecane 0.91 1958 palmitic acid 2.50 1994 1-eicosene 0.70 2000 eicosane 0.77 2100 heneicosane 2.67 2127 methyl linoleate 1.18 2200 docosane 0.73 2300 tricosane 0.88 2357 oleic acid amide 3.23 2433 docosanal 0.68 2494 1-docosanol 2.99 2600 hexacosane 0.41 2700 heptacosane 0.56 2816 (E,E,E)-squalene 0.45 2900 nonacosane 1.19 Total Identified (%) 97.89 Compounds Identified 57 a RI determined with respect to a homologous series of n-alkanes on an HP-5ms column b 50% (+)- -pinene / 50% (–)- -pinene. c 100% (+)-limonene. d Unidentified:α MS fragmentationα of unidentified compound in Appendix e 35% (+)-linalool / 65% (–)-linalool. f 30% (+)- -terpineol / 70% (–)- -terpineol. g Compounds in bold indicate they are the major components of the essential oil. α α

Compounds present in larger than 3% composition were considered

major compounds. These compounds are all six-carbon fatty acid derivatives

from the C. canadensis bark oil. They include 1-hexanol (23.33%), hexanoic

acid (18.15%), (2E)-hexenoic acid (3.44%), oleic acid amide (3.23%), and

1-docosanol (2.99%)-whose structures are shown in Figure 19 below.

H C OH a) 3 b) H3C OH

O NH2

CH c) H3C OH d) 3

H3C OH e) 20

Figure 19: Major volatile compounds of Cercis canadensis bark: a) 1-hexanol, b) hexanoic acid, c) (2E)-hexenoic acid, d) oleic acid amide, and e) 1-docosanol

3.2 Compounds Identified from Zanthoxylum clava-herculis

The following compounds were isolated from Z. clava-herculis bark essential oil extraction. Volatile compounds from the essential oil of the bark were identified using GC-MS spectra.

3.2.1 Zanthoxylum clava-herculis Essential Oil

The bark was collected for hydrodistillation on 13 November, 2016.

From the original mass 146.01 g of chopped bark, 4.24 g essential oil was

collected. The oil was collected then stored in a new scintillation vial and was

clear and colorless, as shown in Figure 20 below.

Figure 20: Essential oil from the bark of Z. clava-herculis

The data collected from the GC-MS was analyzed and 66 compounds

were identified in the bark essential oil. The enantiomeric distribution using

chiral gas chromatography-mass spectrometry is given for the

monoterpenoids. The chirality is given below Table 4. The compounds and

their composition percent are given in Table 4 below.

Table 4: Essential oil of Zanthoxylum clava-herculis Bark Oil

RIa Compound %

769 3-methyl-2-buten-1-ol 0.03 782 3-methyl-2 butenal 0.03 801 hexanal 0.03 864 1-hexanol 0.03 925 -thujene 0.82 933 -pinene 1.85b 949 campheneα 0.04 973 sαabinene 46.32c 978 -pinene 2.39b 989 myrcene 1.54 1007 -phellandreneβ 0.06 1017 -terpinene 2.63 1025 α p-cymene 0.24 1030 αlimonene 18.39d 1031 -phellandrene 0.39e 1032 1,8-cineole 0.26 1035 β(Z)- -ocimene 0.02 1046 (E)- -ocimene 0.05 1049 oβ-cresol 0.05 1058 γ-terpineneβ 3.96 1070 cis-sabinene hydrate 1.38 1085 terpinolene 0.92 1090 p-cymenene 0.02 1100 linalool 0.17f 1101 trans-sabinene hydrate 1.20 1105 1-nonanal 0.03 1113 1,3,8-p-menthatriene 0.02 1118 trans-thujone 0.03 1124 cis-p-menth-2-en-1-ol 0.73 1142 trans-p-menth-2-en-1-ol 0.45 1150 verbenol 0.01 1178 lavandulol 0.03 1181 terpinen-4-ol 12.76g 1195 -terpineol 0.51h 1196 cis-piperitol 0.17 1206 (2E)-αoctenol acetate 0.02 1208 trans-piperitol 0.26 1218 β-cyclocitral 0.02 1223 cis-chrysanthenyl acetate 0.08 1226 citronellol 0.05

RIa Compound %

1292 2-undecanone 0.03 1297 carvacrol 0.02 1308 p-vinyl-guaiacol 0.09 1339 methyl-anthranilate 0.04 1350 eugenol 0.15 1375 -copaene 0.01 1377 geranyl acetate 0.02 1389 α-elemene 0.02 1399 methyleugenol 0.02 1405 methyl N-βmethyl anthranilate 0.04 1419 -caryophyllene 0.26 1455 -humulene 0.04 1481 βgermacrene D 0.51 1495 bicyclogermacreneα 0.09 1518 -cadinene 0.03 1548 elemol 0.05 1576 spathulenolδ 0.03 1582 juneol 0.09 1593 methoxy-eugenol 0.03 1655 -cadinol 0.03 1684 germacra-4(15),5,10(14)-trien-1 -ol 0.03 1714 (2Eα,2Z)-farnesol 0.12 1736 (2E,6E)-farnesol α 0.02 1965 sandaracopimara-8(14),15-diene 0.06 2161 neoherculin 0.12 2186 herculin 0.02 Total Identified (%) 100 Compounds Identified 66 a RI determined with respect to a homologous series of n-alkanes on an HP-5ms column b 98% (-)- -pinene/ 98% (-)- -pinene c 95% (-)-sabinene d 99% (+)-αlimonene β e 98% (-)- -phellandrene f 98% (-)-linalool g25% (+)-βterpinen-4-ol/ 75% (-)-terpinen-4-ol h55% (-)- -terpineol/ 45% (+)- -terpineol i Compounds in bold indicate they are the major components of the essential oil. α α

Compounds present in larger than 3% composition were considered

major compounds. These compounds and their enantiomeric distribution

from the Z. clava-herculis bark oil were 95% (-)-sabinene and 5% (+)-

sabinene (46.32%), (+)-limonene (18.39%), γ-terpinene (3.96%), and 25% (+)-

terpinen-4-ol and 75% (-)-terpinen-4-ol (12.76%), shown in Figure 21 below.

CH3 CH3 CH3 CH3

H3C H3C

a) CH2 CH2 b) H2C CH3 H3C CH2

CH3 CH3 CH3

H3C OH H3C OH c) CH3 CH3 d) H3C CH3

Figure 21: Major compounds of Z. clava-herculis bark: a) (+)-sabinene on the left and (-)-sabinene on the right, b) (+)-limonene on the left and (-)-limonene on the right, c) (+)-terpinen-4-ol on the left and (-)-terpinen-4-ol, and d) -terpinene

CHAPTER FOUR

DISCUSSION

4.1 Compounds from Cercis canadensis

The bark of C. canadensis was studied for identification of both nonvolatile and volatile components. The bioactivities of some of the compounds identified have been reported in previous studies discussed below.

4.1.1 Nonvolatile Compound: Lupeol

The compound identified through the extraction of C. canadensis bark was lupeol. Lupeol is a pentacyclic triterpene with properties similar to the non- steroidal anti-inflammatory, indomethacin58. It showed reduced swelling in rats with administration of 50 mg/ kg lupeol58. In addition to its anti-inflammatory properties, lupeol has other medicinal properties for potentially treating other ailments. One study found lupeol to be a potential treatment for acne vulgaris59. It has been shown to be particularly beneficial to the pancreas for treating acute

pancreatitis60. It also has been tested in MTT assay against human pancreatic

adenocarcinoma cells AsPC-1, and found to inhibit their growth, leading to the

assumption of its possible benefit against pancreatic cancer 61.

Lupeol is a compound present in many fruits, especially mangoes, Mangifera

indica, from the family Anacardiaceae1,62. It has proven antioxidant effects63. Its

anticancer properties have been found to apply to more than just pancreatic cancer.

Lupeol has shown inhibitory properties against types of bladder, canine oral, and

hepatocellular cancer64–66. The growth of a few other tumor cell lines has been

inhibited by lupeol; these include Hep-G2, A-431, and H4IIE by inhibiting topoisomerase II67. Banana flowers, Musa sp var. Nanjangud rasa bale, from the

family Musaceae contain lupeol and show antihyperglycaemic properties due to its

inhibition -glucosidase1,68. The enzyme -glucosidase is found in the human intestine andof α its inhibition slows down the adsorptionα of carbohydrates69. The

inhibition of -glucosidase can slow down the symptoms of diabetes such as

nephropathyα and neuropathy69.

One plant used in Latin America for inflammation, Angelonia angustifolia,

from the family Plantaginaceae, has proved to have rather high concentration of

lupeol, with the roots containing 9.14 mg/g and the aerial parts of the plant

containing 1.3 mg/g lupeol1,70. This is much greater than the concentration of lupeol

in the bark of C. canadensis, which was 0.012 mg/g lupeol in dried, chopped bark.

Lupeol is very common in many families of plants. In addition to the three mentioned above, it is seen in many legumes other than the C. canadensis. It has

been found to be present in the legumes Acacia raddiana, Lotus japonicus roots,

Alhagi maurorum, and others71–73. The legume A. maurorum has an unusually high

concentration of this bioactive compound, with a 0.11 wt % in the plant’s root bark,

which also has recorded anti-inflammatory use73.

Although the C. canadensis bark contained little lupeol from the crystallization in this experiment in comparison to the A. angustifolia and

A. maurorum, the bark has some similar recorded properties to these plants. C. canadensis bark’s use against whooping cough suggests its use as a treatment of inflammation. The inflammation of the respiratory tract caused by the bacteria B. pertussis is the reported cause of whooping cough10. The presence of lupeol in C.

canadensis bark could be one chemical reason it was used to treat whooping cough.

4.1.2 Volatile Components of Cercis canadensis Bark

The main volatile constituents of the C. canadensis bark, which were present

in greater than 3% abundance in the bark oil extract, were 1-hexanol (23.33%),

hexanoic acid (18.15%), (2E)-hexenoic acid (3.44%), oleic acid amide (3.23%), and

1-docosanol (2.99%). These are all fatty acids or fatty acid-derived alcohols and aldehydes, which are seen in the essential oils of other plants of Fabaceae. The bark of Cassia bakeriana from Brazil of the Fabaceae family revealed the presence of

51.3% fatty acids, 23.2% aldehydes, and 11.1% alcohols in its essential oil74. The

bark essential oil of another Fabaceae plant, Inga laurina from Brazil, was composed

of 46.8% fatty acids75. Another member of the Fabaceae, Robinia pseudoacacia,

growing in Poland has a composition of 65.1% aliphatic alcohols from its leaf essential oil76.

The first main volatile compound, 1-hexanol, which made up 23.33% of C. canadensis bark oil, is found in black tea from Yichang City, Hubei Province77. Black

tea has numerous proven health benefits, including lowering cholesterol, decreasing

the occurrence of digestive and urinary tract cancer in postmenopausal women, and

decreasing the storage of slow-release carbohydrate sources78–80. The compound 1-

hexanol is also found in various species of cherries, including various cultivars of

Prunus avium; it has been found in large composition from 45.871 to 72.663% in

nine commercial cherry wines made by the Shandong Zunhuang Cherry Wine Co.

made from sweet cherries, Cerasus pseudocerasus81,82. The health benefits of

cherries, Prunus avium, include antioxidant activity and the ability to scavenge

radicals83,84. The six-carbon fatty acid 1-hexanol can be found in other natural

products.

Another natural source of 1-hexanol is the fruit of olive trees, Olea europea;

1-hexanol has been identified in 39 virgin olive oil samples85. Virgin olive oil has

shown natural anti-inflammatory effects due to the presence of the component

oleocanthal, which is structurally similar to ibuprofen86. The compound 1-hexanol

also occurs in green leaves and shows antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, and Salmonella enteritidis87.

Data suggest that longer chain fatty alcohols show greater antimycobacterial activity

with chains of seven to ten carbons88.

Hexanoic acid is the next major volatile constituent of the C. canadensis bark

oil, at 18.15% of the oil; this compound has proven antifungal activity. The fungus

Alternaria alternata, which affects the growth of plants, was resisted by treating

plants with hexanoic acid89. The use of hexanoic acid as a fungicide could extend to

resisting Pseudomonas syringae, which is a pathogen that commonly affects tomato plants. Hexanoic acid could work as a fungicide by countering the impact of coronatine and jasmonyl-isoleucine on the salicylic acid cycle pathway90. Hexanoic

acid, along with other fatty acids, has exhibited inhibition of Streptococcus mutans, S.

gordonii, S. sanguis, Aggregatibacter actinomycetemcomitans, Fusobacterium

nucleatum, and Porphyromonas gingivalis91. Hexanoic acid showed better inhibition

against Candida albicans than other fatty acids with shorter carbon chains that were

also screened91.

A biosynthetic pathway for the synthesis of hexanoic acid has been

constructed in yeast, Kluyveromyces marxianus, from galactose with the use of 5 to 7 genes cloned from various sources; the most stable pathway found was through a chain elongation using the malonyl CoA-acyl carrier protein transacylase from

Saccharomyces cerevisiae92. Another process of making hexanoic acid that has been

studied to determine optimized production is extractive fermentation from the

compound galactitol within Clostridium sp. BS-1, which naturally produces hexanoic

acid as a metabolite93. This hexanoic acid formation has been pursued because it

could be the end product of a reaction producing bioenergy. This reaction converts

D- and L-galactose to galactitol, which is then utilized by Clostridium as a

fermentable carbon source forming hexanoic acid94. At the time of this study, the

metabolic pathway of hexanoic acid production in Clostridium had not been

determined94. The metabolic pathway of an anaerobe, Megasphaera sp. MH, that also

produces hexanoic acid was studied later, finding that hexanoic acid is produced

through the metabolizing of fructose, which can be increased by adding acetate to

the medium95.

The compound 2E-hexenoic acid has the smaller composition in the C.

canadensis bark oil of 3.44%. It has been found in small percentages in some plants

with medicinal uses. One such plant is the wood apple (Feronia limonia), whose fruit pulp contains 2E-hexenoic acid. The F. limonia has displayed antioxidant and antimicrobial activity against Staphylococcus aureus and Bacillus cereus96. Oleic acid

amide, also known as oleamide, has been found to make up 3.23% of the C.

canadensis bark oil. Oleamide can be formed from oleic acid and ammonia in an

enzyme-catalyzed reaction in the brains of mammals. Ammonia is produced and

oleamide is detected in sleep-deprived animals-specifically in the cerebrospinal fluid, but not in well-rested animals97,98. Oleamide shows hypnogenic activity that is

independent of changes in the mammal’s blood pressure, heart rate, or body

temperature99. The production of oleamide has also been observed with the

reactants oleic acid and glutamine, although the mechanism of oleamide’s formation

is unknown98.

The last major volatile compound found in the C. canadensis bark oil is 1-

docosanol, making up exactly 3% of the oil found. This compound has shown

antiviral activity against various viruses, including wild-type herpes simplex,

acyclovir-resistant herpes simplex, and respiratory syncytial virus100. The suggested

mechanism of inhibition is the interruption of the virus entering the target cell100. It

has also been found in small percentages in the outer bark of Pinus densiflora101.

This compound has been found in the essential oil of two mosses from Northern

Europe, Rhytidiadelphus triquetrus and Polytrichum commune; the first moss showed

activity against E. coli, while the second moss, which had a higher concentration of

1-docosanol in comparison to the first moss, R. triquetrus, showed activity against

Bacillus cereus and Staphylococcus aureus102.

The enantiomeric distribution of monoterpenes in C. canadensis found a

racemic mixture of 50% (+)- -pinene, 50% (–)- -pinene, that (+)-limonene dominated, while there was 35%α (+)-linalool andα 65% (–)-linalool, and 30% (+)- - terpineol with 70% (–)- -terpineol. The essential oil of mandarin has a similar α enantiomeric distributionα for almost all of the monoterpenes studied, showing an almost -pinene with 54% (+) and 46% (-), 98% (+)- limoneneracemic, and 27 distribution% (+)- -terpineol of α and 73% (–)- -terpineol103. The plant Salvia

viridis L. from the Lamiaceaeα family contains a similarα enantiomeric distribution of

linalool to C. canadensis, displaying a distribution of 32.4% (+) and 67.6% (-); its essential oil is composed of 12.7% linalool104.

4.2 Compounds from Zanthoxylum clava-herculis

The main volatile constituents of the Zanthoxylum clava-herculis bark oil extract- which were present in the oil- were 95% (-)-sabinene and 5% (+)-sabinene

(46.32%), (+)-limonene (18.39%), γ-terpinene (3.96%), and 25% (+)-terpinen-4-ol

and 75% (-)-terpinen-4-ol (12.76%). Compared to the essential oil composition of

the bark of the Z. clava-herculis, the leaf oil was dominated by limonene (43.6-

73.0%) and 1,8-cineole (12.9-43.3%), with trace amounts of sabinene and terpinen-

4-ol21. The bark essential oil of Z. tetraspermum also did not show the major

components of Z. clava-herculis; however, it had the major component of

caryophyllene oxide (70.95%)105. The Z. clava-herculis bark has a unique composition among other Zanthoxylum plants.

Sabinene, the main volatile constituent of Z. clava-herculis bark oil, is a monoterpene that is found in other natural sources and could be a component in aircraft fuels in coming years106. The efficient synthesis of sabinene with an

engineered E. coli strain was performed by converting sabinene from glycerol with a

gram-to-gram efficiency of 3.49%. This is a green, renewable source for sabinene

production106. Sabinene is found in other bioactive plants. This compound was the

main constituent of the essential oil found in Cordia verbenacea from the family

Boraginaceae that has anti-inflammatory, analgesic, and anti-ulcerogenic

activities107. Sabinene can be found in the southern European plant Siler montanum

from the family Apiaceae in both of its subspecies, montanum and siculum, ranging

from 4.6 to 17.9% composition of the essential oil from the plant’s dried aerial

parts108. Sabinene has been found in the essential oil of rough lemon leaves, Citrus

jambhiri of the family Rutaceae, in the composition of 5.9%1,109. Sabinene has

specifically shown antifungal activity against Alternaria alternata110.

The enantiomer that dominated the Z. clava-herculis bark oil was (-)- sabinene, making up 95% of the compound obtained. The (-) enantiomers also dominated the cis- and trans-sabinene hydrate found in the bark oil. The enantiomer

(-)-sabinene has been found to dominate bergamot (Citrus bergamia) oil from 81.2 to 85.9% composition111; however, in mandarin (Citrus reticulata) oil, the (+)

enantiomer dominates112,113. The (-)-sabinene is a major component of the essential oil of the aerial parts of Aloysia sellowii, which has antimicrobial activity against

Gram-positive and Gram-negative microorganisms and two yeasts114. The

dominance of this enantiomer as one of the major constituents of A. sellowii, leads to the conclusion that (-)-sabinene has some responsibility for this bioactivity and could be responsible for the activity seen in the medicinal use of Z. clava-herculis by the Houma and Cherokee Indians114.

Another main component of the Z. clava-herculis was limonene, which has been previously found in the Z. clava-herculis leaf essential oil21. Similar to the

composition seen in Z. clava-herculis bark, limonene has been found in many plants

where sabinene is found, including Siler montanum and Citrus jambhiri 108,109. The

Citrus jambhiri leaves contain a 46.5% essential oil composition of limonene109.

The (+)-limonene enantiomer dominated the Z. clava-herculis bark oil, and

has been found to be the dominant enantiomer in bergamot, mandarin oils,

grapefruit (Citrus paradisi), lemon (C. medica), lime (C. aurantifolia), orange (C. sinesis), and kumquat (Fortunella margerita)111–113,115. The (-)-limonene enantiomer dominates white pine emissions, Pinus strobus L., the xylem of Picea abies, and the needles of Pinus sylvestris116–118. The (-) enantiomer also shows 83% composition of

the limonene found in Zanthoxylum schinifolium fruits119. From the same family, Z.

hyemale exhibits a racemic mixture of both limonene enantiomers in its fruit and

flowers120.

The major compound γ-terpinene is found in Thymus vulgaris from the family

Lamiaceae in a similar percent of 4.58% to that seen in Z. clava-herculis bark

(3.96%)52. This compound has been seen to make up 20.65% of tea tree, Melaleuca

alternifolia, oil, which inhibits the growth of human melanoma M14 cells121. This

monoterpene has also been found to slow the peroxidation of linoleic acid, which

has potential applications in foods with lipids by increasing their shelf life122. This

compound has not been reported as a major component of many plant essential oils.

One plant that has similar percentage to the oil of Z. clava-herculis is the Ficus

hispida female receptive fig, γ-terpinene making up 4.8% of its essential oil123. In

the F. hispida female post-pollinated fig, however, there is no detected γ-terpinene in

the oil123. The change in essential oil composition before and after pollination indicates that γ-terpinene is a compound necessary for attracting the wasp

pollinators to the female figs123. The tree sap of Mangifera indica has γ-terpinene in

some of its varieties up to 2.54% composition; the sap from two mango varieties,

whose essential oil composition was previously determined to contain γ-terpinene,

was screened against various bacteria and fungi124,125. The non-aqueous phase of the

sap from the M. indica variety with greater percent γ-terpinene, showed greater

antifungal properties against Penicillium than the other mango varieties screened125.

This variety also proved to control the spore germination better than the other

variations tested125.

The compound terpinen-4-ol showed an enantiomeric distribution of 25%

(+)-terpinen-4-ol and 75% (-)-terpinen-4-ol and made up 12.76% of the Z. clava-

herculis bark oil. This distribution is seen in mandarin peel essential oils with (-)-

terpinen-4-ol showing a 71.3 to 90% composition112. The opposite ratio is seen in

rosemary (Rosmarinus officinalis L.) oil 73.2 to 75.7% of enantiomer (+)-terpinen-4-

ol126. The enantiomeric distribution in thyme, Thymus vulgaris L., oil was found to

be 70% (+)-terpinen-4-ol52. The aerial parts of another common herb, oregano,

Origanum vulgare, essential oil has almost opposite enantiomeric distribution of

terpinen-4-ol than Z. clava-herculis, but is similar to that of thyme with a

distribution of 78.7% (+) and 14.3% (-)127.

Some other bioactive plants, specifically the antifungal Ocimum canum and O. kilimandscharicum, contain terpinen-4-ol in their essential oils. The O. canum shows a 59.4% (+) and 40.6% (-) enantiomeric distribution, while O. kilimandscharicum shows 84.2% (+) and 15.2% (-)128. Terpinen-4-ol has shown inhibition of the growth of human melanoma cells M14, and can be found in a high percentage (42.35%) in tea tree, Melaleuca alternifolia, oil121. It has been found to suppress the growth of lung cancer cells as well129. The known bioactivity of terpinen-4-ol suggests it has

some responsibility for what activity the Houma and Cherokee Indians saw in the

use of the Z. clava-herculis bark.

The enantiomeric distribution of the non-major monoterpenes of the oil was

also determined. The (-)-enantiomers - -pinene, linalool -

phellandrene displayed a 98% occurrenceof α inpinene, the Z. βclava-herculis oil. andThe β(-)- -

-pinene enantiomers are the major enantiomers found in lemonα peel

(pineneCitrus medicaand β ), lime fruit and peel (C. aurantifolia), and neroli blossom (C. autantium) oil115. Interestingly, the Z. schintfolium was not dominated by the (-)- -

pinene and linalool enantiomers, but by the (+)- -pinene enantiomer119. The (-)-α linalool also dominated the bergamot oils with aβ similar 99% occurrence, while the

(+) enantiomer dominated the mandarin oils with an 80.7 to 87.3%111,112.

-Phellandrene also shows the (-) enantiomer almost exclusively in the species

Pinusβ tropicalis, P. maestrensis, and in the needles of P. sylvestris118,130.

One monoterpene that showed close to a racemic enantiomeric distribution in Z. clava-herculis is the -terpineol with 55% (-) and 45% (+) distribution. The (-)-

-terpineol enantiomer isα found in 68-75% occurrence in mandarin oil, in cinnamon withα 80% occurrence, and in Laurus nobilis in 64-69% occurrence112,131. The (+)- -

terpineol enantiomer was found to have greater occurrence in Micromeria fruticoseα

of 78-88%131. The (+) enantiomer is also found in bergamot oils in 56-79%111.

CHAPTER FIVE

CONCLUSION

The bark of two medicinal shrubs, Cercis canadensis and Zanthoxylum clava-

herculis, were analyzed phytochemically during this project. This study led to the

identification of lupeol in the extraction of nonvolatile compounds and major

volatile compounds 1-hexanol, hexanoic acid, (2E)-hexenoic acid, oleic acid amide,

and 1-docosanal from the essential oil extracted from C. canadensis. Lupeol was

found to make up 0.0007% of the mass of the crude extract. The presence of lupeol and medium and long-chain alcohols, aldehydes, and carboxylic acids could account for the Cercis canadensis medicinal uses by Cherokee and other Native American tribes.

The volatile compounds that were major components of Z. clava-herculis were sabinene, limonene, γ-terpinene, and terpinen-4-ol. Each of these compounds

has been found in other plants with medicinal uses. Their presence clearly has some

relation to the benefits reported from using this bark. It is still unclear, however, if

any of these compounds are solely responsible for the bark of the Z. clava-herculis’

anti-inflammatory property.

Future studies of the Cercis canadensis bark include a polar extraction of

nonvolatile compounds using a solvent such as methanol and identification of these

compounds. The isolated nonvolatile compounds and the essential oil collected from both C. canadensis and the Z. clava-herculis should undergo antimicrobial screening to find each individual compound and essential oil’s antimicrobial activity.

The specific quantity of each bioactive component needs to be identified in

each bark to find out if the amount present would give the biological response

reported by the Cherokee. If any compound from each plant indicates that it is

individually responsible for medicinal properties, its mechanism of biological

activity should be studied. Furthermore, to fully understand the phytochemistry of

both barks, collections and phytochemical analysis should be done at various times

throughout the year. This study suggests an initial chemical reason for the use of

both Cercis canadensis and Zanthoxylum clava-herculis by Native American tribes.

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APPENDIX

Unidentified compound from Cercis canadensis bark oil MS fragmentation with the GC RI 1092:

Figure A: Mass Spectroscopy Fragmentation of Unidentified Compound in Cercis canadensis bark essential oil