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Synergistic Bioactivity Testing of a Medicinal
Herb From the United States Virgin Islands
Jaime Heimbegner, B. S.
2004
Sweet Briar College
Synergistic Bioactivity Testing of Extracts from a Medicinal
Herb of the United States Virgin Islands
A Senior Honors Thesis in the Department of Chemistry
Sweet Briar College
Jaime Lee Heimbegner
Defended and Approved 09 April 2004
Awarded High Honors
C34-
4/- / ^^
Prof. Jomr J. Beck
Thesis Project Faculty Advisor
Date
^:^
Prof. JlJlJsJ. Granger
^
^ .-7 -ff->^ iC^
^7^Ah
Date
f/jAi
Mr. William G. Trankle, Eli Lilly
Date
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
ii
DEDICATION
i"
LIST OF FIGURES
—
iv
LIST OF SCHEMES
v
LIST OF TABLES
vi
ABSTRACT
1
INTRODUCTION
2
RESULTS AND DISCUSSION
1
2
CONCLUSION
-— 26
EXPERIMENTAL
27
FUTURE RESEARCH
32
REFERENCES
33
LIST OF APPENDIX A CONTENTS
36
APPENDIX A
35
LIST OF APPENDIX B CONTENTS
43
APPENDIX B
45
~ ACKNOWLEDGEMENTS ~
The
undertaking
and completion
of an Honors
Thesis
is
a
difficuh
venture
that
is
dependent upon many people.
I would
like to acknowledge just
a few of the many people
involved for their guidance and support along the way. First, my advisor, Dr. John Beck: Thank
you for always being so eager to teach me and for pushing me to do my best. You have done an
excellent job preparing me for my graduate school endeavors and
I know that
I will continue to
make you proud. You
are
an
excellent
teacher and
don't
ever
forget
it! My
parents,
Nita
Heimbegner and Errol Effatt:
I will never be able to thank you two enough for all that you have
done for me and for all of the support that you have given me throughout the years. You have
both been role models and motivating forces in my life, and
I cannot even begin to express how
much you mean to me.
I would also like to thank the facuhy of Sweet Briar College's Chemistry
Department
for always
being
so
supportive and
for helping me
to
realize my passion
for
chemistry.
And,
finally and most
importantly.
I would
like
to
thank
Sweet
Briar
College
(Endeavor's
Grant),
the
National
Science
Foundation MRI
(Grant
#
0216162),
and
the
Commonwealth Health Research Board (Grant # 02-16) for funding my research.
~ DEDICATION ~
I would like to dedicate this work to my family, friends, and teachers who have played
and integral role in its completion.
I could not have made
it through without their knowledge,
guidance, and support. Thanks for always being there for me when I needed you. You are all
much appreciated and 1 will never forget you.
HI
~ LIST OF FIGURES ~
Page
Figure 1.
Aspirin
2
Figure 2. Quinine-
2
Figure 3.
Quinacrine, Chloroquine, Primaquine—
3
Figure 4.
Taxol'^ and Callipeltoside A
5
Figure 5.
Stuctures of known compounds in lemongrass
8
Figure 6.
Penicillin G
9
Figure 7.
Ampicillin
9
Figure 8.
Structure of peptidoglycan
-
10
Figure 9.
Structure of a lipopolysaccharide
1
1
Figure 10.
Generic alkaloid in the (a) N-oxide, (b) free amine,
and (c) protonated forms
14
Figure 11.
Division of hexanes fractions by TLC—
17
Figure 12.
Citral. Linalool, p-Elemene, P-Caryophyllene,
and a-Humulene
21
IV
~ LIST OF SCHEMES ~
Page
Scheme 1.
Extraction of lemongrass
13
Scheme 2. Zinc reduction of alkaloids in N-oxide form
14
~ LIST OF TABLES ~
Page
Table 1. Average zones of inhibition (mm) for lemongrass
crude extract
1
2
Table 2. Average zones of inhibition (mm) for lemongrass
crude layer from the liquid/liquid partition
16
Table 3. Average zones of inhibition (mm) for synergistic testing of lemongrass
crude layers from the liquid/liquid partition (reverse synergy)
16
Table 4. Average zones of inhibition (mm) for hexanes layer fractions
17
Table 5. Average zones of inhibition (mm) for synergistic testing of
the hexanes fractions against B. subtilis
20
Table 6. Average zones of inhibition (mm) for synergistic testing of
the hexanes fractions against S. aureus
20
Table 7. Average zones of inhibition (mm) for the ethyl acetate fractions
23
Table 8. Average zones of inhibition (mm) for synergistic testing of
the ethyl acetate fractions against B .subtilis--
24
Table 9. Average zones of inhibition (mm) for synergistic testing of
the ethyl acetate fractions against S. aureus
25
Synergistic Bioactivity Testing of Extracts from a Medicinal
Herb of the United States Virgin Islands
Abstract:
The
research
herein
reports on
the
antibacterial
activity of the
medicinal
herb
lemongrass {Cymbopogon flexuosus) and possible synergistic
activity between
its extracts and
the known antibiotics penicillin and ampicillin. The novel bioactive compounds of this medicinal
herb are well known, thus this research focuses on confirming the plant's antibacterial activity,
and more importantly, identifying possible synergistic
activity. Disc susceptibility testing was
performed against the bacteria Bacillus subtilis and Staphylococcus aureus to obtain background
and
synergistic bioactivity data.
Critical analysis of the results
led
to the conclusion that an
enhancement
in bioactivity
is exhibited by combining several plant
extracts of Cymbopogon
flexuousus with the known antibiotics penicillin and
ampicillin. However, due to the lack of
consensus pertaining to the issue of drug interaction, there are several equally valid models for
evaluation of drug interaction. Since the definitions of drug synergism critically depend on the
reference model
for
interaction,
this
research
only
reports
the
demonstration
of enhanced
bioactivity.
~ INTRODUCTION ~
Medicinal herbs contain substances known to modem and ancient civilizations for their
healing properties.
For centuries medicinal herbs have been an important source of remedies for
assorted
illnesses and
injuries." Since the beginning of their usage, medicines have advanced
from
the
simple employment
for
treatment
of ailments
to
the
isolation,
identification,
and
synthesis of their active components so that they can be marketed as commercial drugs.
One
example
is the use of willow tree bark as an effective treatment for reducing
O^OH
fever and relieving pain. The evolution of willow bark as a drug began in the
[|^^ ^
eighteenth
century with
the
isolation of the
active component,
salicin, by
Figure 1. Aspinn
Johann Buchner.' Salicin was
subsequently converted
into
salicylic
acid
via hydrolysis and
oxidation, and proved to be a very successful fever reducing agent." The use of salicylic acid,
however, often led to severe gastrointestinal toxicity, which was later overcome when salicylic
acid was converted
into
acetylsalicylic
acid
via
acetylation.'
hi
the mid-nineteenth century,
laboratory synthesis of the compound began and
it was marketed under the trade name aspirin.
Today, aspirin
is the most widely used synthetic drug, with Americans alone consuming eighty
million pills a day."
Another example of the evolution of a drug in this manner is the use
'^
of the bark of cinchona trees
to reduce
fever, relieve pain, and to
induce
uterine
contractions
during
labor."
Cinchona
bark
was
brought
to Europe
from
South America
in
the
mid-seventeenth
century and the active component, quinine, was isolated in the early
nineteenth century by French chemists
J.
B. Caventou and
P.
J.
Pelletier."*
hi
the
mid-nineteenth
century
laboratory
synthesis
of quinine was
achieved by
American chemists R. B. Woodward and W. E. Doering; and before the recent development of
more effective synthetic drugs such as quinacrine, chloroquine, and primaquine (See Figure 3),
quinine was the specific agent in the treatment of malaria.
^0.
^ ^
y^i^^N.
"N" ^^
"CI
^
^
Quinacrine
Chloroquine
Primaquine
Figure 3
Following World War II there was a considerable investment by companies in the search
for new drugs from plants.^ However, after 1960 interest in the search began to fade and by 1974
only one pharmaceutical company in the United States was investigating plant-derived drugs.
By 1980, the total pharmaceutical budget of the U.S. had grown from S723 milhon to S2 billion,
yet not a single U.S. company was researching potential drugs from higher plants." The decrease
in interest can be attributed to the introduction of automation, robotics, and personal computers
to the drug discovery scene. ^
The introduction of this technology made chemistry the
rate-
limiting step in drug discovery programs.^ Less than a decade later, there was a renewed interest
in investigating plants as a likely source of new commercial drugs. One reason for the dramatic
reversal of attitude can be attributed to the successful trials on Taxol* and other plant compounds
that have shown encouraging activity against cancer and diseases such as HIV. Some companies
continue to investigate natural products as a source of life-improving pharmaceuticals.
Bayer,
Merck, and Wyeth are major drug companies that have remained committed to natural product
drug discovery.^ With the current increase of new viruses and diseases, the desire to find new
drugs from plants is at is peak.
In addition to the advent of new illnesses, the importance of new drug discovery fi-om
plants is critical due to the rapid destruction of the world's vegetation, threatening the extinction
of many species.
As our natural resources are being consumed the window of opportunity for
the discovery of new drugs from plants diminishes.
It has been estimated that the number of
plant
species on
Earth
is
well
over
250,000,
of which only
about one percent have been
investigated in any depth in terms of their bioactive potentials." With many active compounds
yet to be discovered and fully evaluated, the investigation of natural resources in an effort to
discover, manipulate, and synthesize new medicine, is of great importance to the pharmaceutical
industry.
It
is estimated that twenty-five percent of prescription drugs contain plant-derived
active ingredients and an even greater percentage is based on semi -synthetic or wholly synthetic
ingredients originally isolated fi-om plants.' According to a recent survey by the National Cancer
Institute, 61%
of
the
877
small-molecule new
chemical
compounds
introduced
as
drugs
worldwide during 1982-2002 can be traced to or were inspired by natural products.
In 1985
2,618 new structures were isolated from plants, most of which were beyond the imagination of
the most inventive chemists."
Molecules such
as Taxol® and callipeltoside A (Figure 4) are
prime examples of compounds whose
structural complexity
still manage
to keep chemists
in
awe.
In addition
to
their complex
structures
these compounds
are
also of great
interest
to
chemists because of the cytotoxic activity they exhibit against cancer cells.
AcO
p
OH
Ph
NH
O
OHE
OAc
OBz
CI
Taxol''
Callipeltoside A
Figure 4
Many chemists have been eager to research medicinal plants that
exhibit antibacterial
activity due to the recent health problem of increased bacterial resistance to antibiotics.
Despite
the fact that antibiotics dramatically transformed medical care by reducing illness and death from
infectious diseases, the bacteria that they inhibit have developed a resistance to them over the
decades.
Excessive and inappropriate use of antibiotics promote this resistance, which occurs
when the bacteria change in some way that reduces or eliminates the effectiveness of drugs or
other
agents designed
to
cure
or prevent
infections.^ As
a
result of the widespread use of
antibiotics, virtually
all common bacterial infections
in the United
States, and throughout the
world, are becoming resistant towards treatment, and for this reason antibiotic resistance has
become one of the world 's most pressing public health problems.
In order to help combat antibiotic resistance, chemists have started to take a great deal of
interest in drug synergism. Drug synergism is generally defined as the pharmacological effect of
a drug combination
that
is
greater than the
effect of either agent
alone.
Because no
clear
consensus exists relative to the issue of drug interaction, there are several equally valid models
for the evaluation of drug interaction.'^ As a consequence there are several definitions of what
type of drug interaction constitutes drug synergism.
However, there
are two more common
definitions
of drug
synergism; one
states
that drug
synergism
occurs when two
drugs
are
combined so that the action of one drug aids or enhances the action of another.
Using this
definition, the effect of the drug combination does not need to exceed the sum of the individual
effects in order to be considered synergistic but must only be greater than the effect of either
agent alone.
The second definition states that drug synergism occurs when the combined effect
of two drugs exceeds the sum of their individual effects. '° By this definition, in order for a drug
combination to be considered synergistic the effect of the combination must exceed the sum of
the
individual
effects.
Regardless of the reference model of drug
interaction
used,
a prime
example of drug synergism
is the enhancement
that occurs when certain drugs, such
as
the
opiates
codeine
and
morphine,
are
consumed
with
alcohol.
The
effect
produced by
the
combination of the two
is
greater than the
effect produced by
either substance
individually.
Therefore,
in
theory,
drug
synergism
between known
antibiotics
and
plant
extracts
with
antibiotic
activity would allow
for the use of minimal amounts of antibiotics, which
in turn
would result in reduced antibiotic resistance.
ki
order
to
find combinations of drugs
that work
synergistically, chemists
rely on
a
relaUvely new method of testing both known and unknown compounds called synergistic testing.
Synergistic testing examines the bioactivity of a plant extract (or any compound) and determines
if the bioactivity is increased when mixed with a known drug and/or compound. The application
of this method
for the
testing of both known and unknown herbal
extracts not only allows
researchers
to
identify new drug mixtures
for treatment of illnesses and diseases but
also
to
identify combinations of known drugs and natural products that can be particularly effective for
combating bacteria that have become resistant to known antibiotics.
An example of a product that exhibits drug-drug synergy is the dietary supplement "Fen-
phen."
Fen-phen
is the
off-label combination of the
appetite suppressants fenfluramine and
phentermine.
The term
"off-label"
refers
to the use of a drug
for purposes not
specifically
approved by the Food and Drug Administration's (FDA). The synergism of this combination of
drugs results in the suppression of appetite and body weight, the reduction of brain serotonin
levels, pulmonary vasoconstriction, and heart valve disease." The rationale for this combination
of drugs was that they exerted independent actions on brain satiety mechanisms so that
it was
possible
to use lower dosages of each drug and
yet
retain a common
action on suppressing
appetite while minimizing adverse drug effects.
In an effort to contribute to the research and discovery of active components of plants and
their
pharmaceutical
uses,
the
research
herein
investigated
a
plant
that
has
demonstrated
medicinal properties and performed synergistic
testing with
its bioactive components and the
known antibiotics penicillin and ampicillin. A search of the chemical
literature indicates that
synergistic activity between plant extracts has been investigated^" but no literature references on
possible synergistic activity between plant extracts and known pharmaceuticals were found. In
an investigation of multi-drug resistance pumps, Stermitz
et.
al. found that two plant-derived
compounds that do not exhibit bioactivity by themselves potentiated growth inhibitory activity of
the
natural
antibacterial
alkaloid
berberine.'"^
This
discovery by
Stermitz
et.
al,
and
the
promising
results
of the
synergistic
testing
discussed
in
this
thesis,
are
significant
to
the
pharmaceutical industry because they have opened the door for the discovery of a new category
of drugs that can be used to combat the ever-increasing threat of antibiotic resistance.
The synergistic testing performed in this investigation involved a plant commonly known
as lemongrass (Cymbopogon
flexuosus).
Lemongrass
is
a medicinal herb from
the
family
Gramineae that is widely cultivated in the tropics and subtropics.'"*
It has exhibited antimicrobial
activity in previous research'" and has been used to treat a variety of conditions such as acne and
flatulence.'^ Lemongrass
oil has been found to be an important source of citral
(1
in Figure 5.)
which is used for the production of ionones and vitamin A.'^ The chemical literature reports that
this
family of plants has
a history of containing compounds
that could
explain the
plants'
purported bioactivities and in a few instances the structures for known compounds are identified
(see Figxire
5).
Due to the extensive research performed by others
to characterize the novel
bioactive compounds of lemongrass, this research
is able to investigate the plant's antibacterial
activity and more importantly possible synergistic activity with known antibiotics.
la Citral a/ Geranial (Z-isomer)
lb Citral b/ Neral (£-isomer)
O
^O
2 Geraniol
3 Citronellol
4 Citronellyl acetate
0.
^0^
6 Isoeugenol ether
7 Methyl eugenol ether
Figure 5
H
The two known antibiotics combined with the plant extracts for
r J
o
.X-N^V^
synergistic
testing
in
this
investigation
were
penicilhn
and
//
ampiciUin.
Penicillin was used
as one of the co-biotics
in an
Figure 6.
Penicillin G
effort
to
provide
possible
alternate
drug
therapy
treatment
to
patients with mild reactions to penicillin. The first drug-quality penicillin was produced in 1940
and
has
since become
the drug of choice
for most common
NH2
^
bacterial
infections
due
to
its
liigh
activity
rate
and
lack
of
^^:^~-.^^^-K.^H^
s
IT X
toxicity.'^
Due
to
the
specificity of penicillin's
bioactivity,
^==5^
° o'^^^^^'^
I
ampiciUin was used as a second co-biotic in order to provide a
HO
Figure 7. AmpiciUin
broader
range
of
activity.'*^
AmpiciUin
is
a
semi-synthetic
antibiotic and a member of the beta-lactam family.
It
is active against
all penicillin-sensitive
bacteria and has shown to be a versatile and relatively well-tolerated antibiotic.
Both penicillin
and ampiciUin hinder the cell wall synthesis of sensitive bacteria; hence they are classified as
bactericidal.
'
The synergy of the plant extracts and the known antibiotics were evaluated by testing
their bioactivity against bacteria plated on agar.
The strains of bacteria used were B. subtilis and
Staphylococcus aureus.
B. subtilis
is a gram-positive, rod-shaped bacterium found in soil and
decomposing plant residue.'*^
It is a non-pathogenic bacterium to humans, animals, and plants. S.
aureus
is
also
a
gram-positive
bacterium,
but,
unlike
B.
subtilis,
it
is
a
spherical-shaped
bacterium, and is commonly found on the skin or in the mucous membranes of healthy people.
It can cause minor skin infections and food poisoning but can also cause serious and sometimes
fatal infections such as bloodstream infections, surgical wound infections, and pneumonia.""
Bacteria are classified as either gram-positive or gram-negative.
These two main groups
of bacteria can be distinguished through a staining technique devised
in 1884 by the Danish
physician Hans Christian Joachim Gram. Gram found that when different types of bacteria were
stained with
aniline dye methyl
violet followed by an iodine
solution,
the bacteria could be
divided into two groups according to whether or not the addition of alcohol removed the dye
(Gram-negative group) or remained fixed to the cells (Gram-positive group)
Gram-positive
bacteria,
such
as those used
in
this
investigation,
are characterized as
having a
cell wall
structure comprised mainly of peptidoglycan.^'* Peptidoglycan
is a unique
polymer that provides much of the strength and rigidity possessed by bacterial cell walls.
It is a
linear polymer with a backbone that consists of ahemating subunits of N-acetyl glucosamine
(NAG) and N-acetyl muramic acid (NAM).
Attached to each NAM subunit
is a side chain of
four amino acids. ^" Gram-positive bacteria do not possess an outer membrane.
NAM
T T
0H-. .o^^
i
-A
•
c)
V"
r^
b,
OH
I
''v^
NH
MH
J
L_o
NAG= X-aceri gkicosaiiiiiie
i^H
o
XArvI=N'-acer.-lmuraniic add
On
M'OH
OH
NH
1
1
^
^OH
Figure 8. Structure of peptidoglycan
10
Gram-negative
bacteria
are
characterized
as
having
a
cell
wall
structure comprised
mainly of lipopolysaccharides.^'*Lipopolysaccharides are large, complex molecules that contain
both lipids and carbohydrates.^' The
cell wall of gram-negative bacteria
is a thinner structure
than
that
of gram-positive
bacteria,
with
an
outer
layer
that
is more
like
a
cytoplasmic
membrane.^'*
Lipid A
HN
HN
0-antigen
repeat 40 units
Core pdysaccharide
Disaccharjde
diphosphate
Fatty acids
Figure 9.
Structure of a lipopolysaccharide
11
~ RESULTS AND DISCUSSION ~
Due to the humid chmate of the United States Virgin Islands (USVI), the plant material
of lemongrass (Cymbopogon flexiwsus) was oven-dried (40.5
°C),
as opposed
to
air-dried,
to
prevent molding. Once received, the oven-dried plant material was extracted via the 3 x 24 h
extraction method with methanol and concentrated in vacuo.
The crude extract was tested for
bioactivity and exhibited antibacterial activity against the two bacteria to be used for the entire
investigation. Bacillus subtilis and Staphylococcus aureus.
The bioassays were duplicated and
the averages of the zones of inhibition are shown in Table
1
. All replications can be found in
Appendix A-01. As the data in Table
1 shows, the crude extract of lemongi-ass was only partially
active against the two bacteria.
S. aureus was observed to be more susceptible to the crude plant
extract than
B.
subtilis based on
the
larger zones of inhibition
exhibited.
Previous
research
investigating
the
antibacterial
activity of lemongrass
supports
these
results."
The chemical
literature not only shows that lemongrass exhibits antibacterial activity against the two bacteria,
but
it also shows that S. aureus is more susceptible to the plant extract.
Table 1. Average zones of inhibition (mm) for lemongrass crude extract
Bacillus subtilis
Staphylococcus aureus
\
Blank
Penicillin
(Ctrl)
lOfig
lOOjig
lOOO^g
Blank
Penicillin
(Ctrl)
lOjig
loo^g
lOOO^ig
31.3
*4
8.5
32.8
8.5
12
* Average of 8 mm and
mm because one of the discs fell off during testing
A portion of the crude
extract was
subjected
to an acid/base
extraction, and another
portion was
subjected
to
a
liquid/liquid
extraction.
The process used
for the
extraction of
lemongrass
is shown in Scheme
1. The acid/base extraction yielded two
fractions, an organic
layer
collected under
acidic aqueous
conditions and an organic
layer
collected under
basic
aqueous conditions.
12
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J
-
Organic
Layer
Under
Basic
^
Conditions
Acid/Base
Extraction —
-
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Organic
Layer
Under
Acidic
I
Conditions
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13
Despite no literature being found that suggested the presence of alkaloids in Cymbopogon
flexuosus,
alkaloids have been found
in
other
plants of the Cymbopogon
genera."
For
this
reason, the acid/base extraction was performed to rule out the possibility of alkaloid content in C.
flexuosus. Alkaloids can occur naturally in either the N-oxide or free amine form.'^ Examples of
these two forms and the protonated form are given in Figure 10.
R'
R
R'
R
R'
I
I
C^
H
a
b
c
Figure 10. Genenc alkaloid in the (a) N-oxide, (b) free amme, and (c) protonated forms
When
brought
under
acidic
conditions by
addition
of hydrochloric
acid
(HCl),
all
alkaloids in the free amine form are protonated and reside in the aqueous layer. Upon addition of
zinc dust, alkaloids in the N-oxide form are reduced to free amines, and subsequently protonated,
so that they too can be moved to the aqueous layer (see Scheme 2). The addition of zinc dust is
an integral step
in the extraction process because only the alkaloids present in the free amine
form can be extracted; any alkaloids left in the N-oxide form would remain in the aqueous layer.
All other compounds of non-alkaloidal nature would remain in the organic layer during acidic
aqueous
conditions. Ammonium hvdroxide
is added
to
R
R"
R
R'
the solution to deprotonate the alkaloids to the free amine
/^^i^^\
Zn
form, causmg them to move back mto the organic layer
I
"^3'-'
i
^
03
H
for collection. Thin layer chromatography (TLC) of the
Scheme 2. Zmc reduction
organic
layer
extracted under
acidic
conditions and
the
organic
layer extracted under basic
14
conditions indicated that no alkaloids were present
in
C. flexuosus as viewed under UV
light
(254 nm) and vanillin developing spray. lodoplatinic acid would have been better to use as a
developing spray but was not utilized because
it was not available at the time.
The
liquid/liquid
extraction yielded three
fractions,
a hexanes
layer,
an
ethyl
acetate
layer, and a methanol/water layer. As anticipated, the TLC of the organic layer extracted under
acidic conditions during the acid/base extraction was similar to the TLC of the crude hexanes
layer from the liquid/liquid partition, hi addition, the TLC of the organic layer extracted under
basic conditions during the acid/base extraction was similar to the TLC of the ethyl acetate layer
from the liquid/liquid partition.
Despite that different eluent systems were used to obtain the
LLC's, the hexanes and ethyl acetate layers appeared by TLC to contain the same compounds
found in the organic layers obtained from the acid/base extraction.
As a result, the acid/base
fractions were
set aside and only the fractions obtained from the
liquid/liquid partition were
subjected
to
bioactivity
testing. A well-separated TLC
of the methanol
layer could
not be
obtained despite numerous solvent systems used, so
it was also subjected to bioactivity testing.
The results of the bioactivity testing of the liquid/liquid partition fractions are as follows:
The crude hexanes layer exhibited moderate bioactivity only against B. subtilis at concentrations
of 100 and
1000 ^g;
the crude ethyl acetate layer exhibited bioactivity against B.
subtilis
at
concentrations of
1 00 and
1 000
|j,g and against S. aureus at a concentration of
1 000
^ig; and the
methanol/water layer did not exhibit any bioactivity against either bacteria.
All bioassays were
performed in triplicate and the averages of the results are provided in Table 2. All replications
can be found in Appendix A-02.
Because the crude hexanes and ethyl acetate layers exhibited
bioactivity they were subjected
to
synergistic testing with
penicillin.
Interestingly,
for both
samples,
the
greater
the amount of plant
extract
loaded
onto
the
penicillin
disc
the more
15
significant
the decrease
exhibited
in
bioactivity.
This type of behavior was termed "reverse
synergy" due to
its apparent reversal of bioactivity. The bioassays were performed in triphcate
and the averages of the results are provided in Table 3. All replications can be found in Appendix
A-03.
Table 2. Average zones of inhibition (mm) of lemongrass crude layers
from the liquid/liquid partition
Bacillus subtilis
Staphylococcus aureus
Fraction
Blank
Penicillin
(Ctrl)
10
100
1000
fig
Blank
Penicillin
(Ctrl)
10
100
1000
US
Hexanes
26
8.6
10
33.5
Ethvl acetate
33
6.6
11.6
34.6
12.2
Methanol/HiO
33.7
32.3
Table 3. Average zones of inhibition (mm) for synergistic testing of lemongrass crude layers
from the liquid/liquid partition (reverse synergy)
Bacillus subtilis
Staphylococcus aureus
Fraction
Blank
Penicillin
(Ctrl)
Pen./
100 ng
Pen./
1000 fig
Blank
Penicillin
(Ctrl)
Pen./
100 fig
Pen./
1000 fig
Hexanes
34.7
34
29.7
34.3
32.7
31.8
Ethyl acetate
33.3
30.3
26.3
32.7
31
28.3
The compounds of the hexanes layer and the ethyl acetate layer were partially separated
via
flash column chromatography then subjected
to
bioactivity
testing.
Due
to
the minimal
amount of product in each fraction obtained from the column of the hexanes layer, the fractions
were
tested
for bioactivity
against only one bacterium.
All
fractions of the hexanes
layer
exhibited
bioactivity
against
S.
aureus
except
for
one,
HI.
The
fractions
that
exhibited
bioactivity are marked in Figure
1 with an asterisk.
Table 4 provides the averages of the results
and
all replications can be found
in Appendix A-04.
It was interesting to note that the crude
hexanes layer did not exhibit bioactivity against S. aureus but almost all of the fractions of the
16
crude hexanes layer column did. This difference in bioactivity
is most likely due to the removal
of extraneous plant material by column chromatography, which
is present
in greater amounts
than
the
bioactive components of the
plant,
or possibly compounds
acting
as
inhibitors
in
combination.
Table 4. Average zones of inhibition (mm) for hexanes layer fractions
Staphylococcus aureus
|
Fraction
Blank
Penicillin (ctrl)
lOfiS
100 fig
1000 tig
Hla
35.5
H2a
35.5
10
8,5
H3a
36
11
12.5
H4
37
12
10
H5
34.5
11
13
H6
35.5
10.5
17
H7
34.5
9
13
H8
38
7.5
11.5
H9
36.5
8
8.5
To sufficiently continue the investigation, a second aliquot of the crude hexanes layer
was subjected to flash column chromatography to obtain more product. The resultant fractions
from these columns were different due to varying fraction collection patterns. Due to limited
product availability, the fractions from the two columns run on the
hexanes
layer were
combined
based
on
the
Rf
values
of the
compounds
by TLC
visualized
by
vanillin
developing
spray.
Fractions H1-H4
were
combined
to
make
fraction
1
(Hlb),
fractions H5 and H6 were combined to make fraction 2 (H2b), and
fractions
H7-H9
were
combined
to
make
fraction
3
(H3b).
OOO
CDO
OO
Essentially,
all spots (3 total) located on the top third of the silica
Figure 11. Division of Fractions
by TLC
plate were combined, all spots (2 total) located in the middle third
17
of the plate were combined, and
all spots (2 total) located in the bottom third of the plate were
combined
(see
Figure
2).
The new
fractions were
loaded
onto
discs
preloaded
with
the
antibiotics penicillin (10 lU) and ampicillin (10 |ig) then subjected to synergistic testing.
Because
the column
fractions
were
only
tested
against
S.
aureus,
analysis
of
the
bioactivity data against B. subtilis was determined from data obtained for the crude hexanes layer
and since S. aureus has proven to be more susceptible to the lemongrass plant extracts than B.
subtilis.
S. aureus has typically been more susceptible to the lemongrass extracts by 2-3 mm of
inhibition, which can be observed
in Tables
1-3.
The
first
fraction (Hlb) demonstrated an
increase in bioactivity against B. subtilis when combined with penicillin or ampicillin, and did
not exhibit an increase or decrease in bioactivity against S. aureus. Table 4 shows that the H3a
fraction, the most bioactive of those combined to make the Hlb fraction, had a zone of inhibition
of 12.5 mm at a concentration of 1000
|j.g. In order for the compound to demonstrate additive
bioactivity with penicillin
it would have to exhibit a zone of inhibition of approximately 43.5
mm.
Since 1000
|j.g of the
first fraction (Hlb) combined with penicillin only had a zone of
inhibition of 36.3 mm against B.
subtilis,
it can be concluded
that additive
activity was not
demonstrated but rather enhanced bioactivity of penicillin, hi terms of deciphering the results to
determine possible
synergistic
activity,
the
results can be
interpreted
in two
different ways
depending on the definition of synergism used as the basis of analysis. If one defines synergistic
activity as the combined effect of two agents exceeding the sum of their individual effects,
then
synergistic
activity was not demonstrated. However,
if one defines synergistic
activity as the
action of one drug aiding or enhancing the action of another, '° then synergistic
activity was
demonstrated. Since the definition of synergism has not been absolutely established as observed
18
from the chemical Hterature,
it will only be suggested that "enhanced bioactivity" was exhibited
when the plant extract was combined with the known antibiotics.
It is interesting to note that when the zones of inhibition were evaluated by the areas and
not by the diameters, there was a clear enhancement in bioactivity that demonstrated more than
additive bioactivity and was congruent with both definitions of synergistic
activity. Using the
formula jrr', the area of bioactivity for the H3a fraction was 122.72 mm" and 754.77 mm' for the
penicillin.
Their sum
is
877.49 mm", which
is
less than
that of the
1034.92 mm'
area of
bioactivity exhibited when they were tested in combination. When tested together the area of
bioactivity was 157.42 mm" greater than the sum of their individual bioactivities, which suggests
that additive and synergistic activity cannot be accurately quantified by linear measurement. The
results of this new method of measurement poses an interesting question as to which method is
the most accurate when evaluating inhibition of bacteria.
The same type of analysis was applied when determining the type of bioactivity the Hlb
fraction demonstrated when combined with ampicillin and tested against B.
subtilis, as well as
when determining the type of bioactivity demonstrated by fractions H2b and H3b. The second
fraction (H2b) exhibited enhanced bioactivity against B. subtilis when combined with penicillin
or ampicillin, and exhibited reverse synergy against S. aureus when combined with penicillin.
This type of drug interaction
is also referred to
as drug antagonism, which occurs when the
pharmacological effect of one drug (agonist)
is reduced by a second drug (antagonist).^ When
combined with ampicillin and tested against
S. aureus,
fraction H2b did not demonstrate any
change in bioactivity. The third fraction (H3b) exhibited enhanced bioactivity against B. subtilis
when
combined
with
penicillin
or
ampicllin,
and
against
S. aureus when
combined
with
penicillin. Reverse synergy was exhibited against
S. aureus when fraction H3b was combined
19
with ampicillin.
Tables 5 and 6 provide the averages of the results and
all replications can be
found in Appendices A-05 and A-06.
Table 5. Average zones of inhibition (nim) for synergistic testing of the hexanes fractions
Bacillus subtilis
Fraction
Blank
Pen.
(Ctrl)
Pen./
lOjig
Pen./
lOO^ig
Pen./
lOOOfig
Blank
Amp.
(Ctrl)
Amp./
10 ng
Amp./
100^g
Amp./
lOOO^g
Hlb
31
30
33
36.3
35.3
30
32.5
39.6
H2b
33
30
35.6
38.7
32
31
34
38
H3b
29.7
31.7
35
40
32
31.7
35.3
39.3
Table 6. Average zones of inhibition (mm) for synergistic testing of the hexanes fractions
Staphylococcus aureus
Fraction
Blank
Pen.
(Ctrl)
Pen./
lO^ig
Pen./
100^g
Pen./
lOOOfig
Blank
Amp.
(Ctrl)
Amp./
10 ^ig
Amp./
lOO^g
Amp./
lOOO^g
Hlb
34
34.3
34
34
31.7
31.7
31.7
31
H2b
33
32.7
32.3
32
30
30
30
30
H3b
32.3
31.7
32
33.7
33
31.7
29.3
29.3
Because there are many taxa oi Cymbopogon flexuosus,
it was not possible to identify the
taxon or the chemical composition of the plant used in this preliminary investigation.'^ A study
of four taxa of C. flexiiosus revealed
that they possess
distinct chemical characteristics when
compared to each other, but when considered together with their differences in morphology, they
warrant recognition as distinct taxa."
The study determined that linalool (8), P-elemene (9), P-
caryophyllene
(10). and a-humulene
(11)
are
the
only
constituents common
to
all
four
C.
flexuosus
taxa, whereas nine constituents
are common
to
tliree of the plants and twenty-one
constituents to only two of the plants.'^ Although the taxa and chemical composition could not
be identified, 'H NMR spectra of several fractions of the hexanes layer bear resemblance to the
20
'H NMR spectrum of citral,
1,
a mixture of the isomers neral and geranial, which have been
found to be bioactive against both B. subtilis and S. aureus (see 1 in Figure 5).^°' ^^ The 'H NMR
spectra of many hexanes
fractions suggest the presence of citral due to vinyl
signals present
between 5-6 ppm and more importantly the aldehydic signal present just before 10 ppm.
HO-^
18
9
10
Figure 12. (1) citral
, (8) linalool, (9) beta-elemene, (10) beta-caryophyllene, (11) alpha-humulene
Previous research comparing extraction methods for marker compounds in the essential
oil
of lemongrass by
gas chromatography
indicated
that
the hexanes
extract
of a
solvent
extraction contained the highest concentration of citral/' The results of the previous research
supports the hypothesis that the hexanes layer of the liquid/liquid partition performed
in this
investigation
contains
citral.
Another
study
that
supports
this
hypothesis
investigated
the
composition of the essential
oil of C. flexuosus,
in which a gas chromatogram of the hexanes
fraction
indicated
the
presence
of a
possible
metabolic
precursor of
citral,
3,7-dimethyl-l-
octene.^^
Proton NMR data of the hexanes fractions also suggest the presence of compounds 8 and
10.
Comparison of the spectra shows signals present
at approximately 6 ppm, indicating the
presence of an internal alkene. These signals have a more complex splitting pattern than those of
21
a terminal alkene, which should be present around 5 ppm.
The terminal alkene signals along
with the other signals could not be identified in the spectra of the hexanes fractions due to the
presence of other signals in the same chemical shift range.
Although the 'H NMR data does not
positively
identify the presence of compound
8
in the hexanes
layer,
the occurrence of the
terminal alkene signal and the fact that the ^H NMR data of the ethyl acetate and methanol layers
do not bear any semblance to the linalool spectrum, provide evidence of compound 8 occurring
in the hexanes layer.
The 'H NMR data also suggests the presence of the compound 10 in the hexanes layer
because of the doublet
at approximately 4.8 ppm, which distinctly indicates the presence of a
terminal alkene.
The other signals could not be distinguished due to the interference of other
signals.
Proton NMR data was unavailable in the chemical literature for compounds 9 and 11,
and as a result their spectra could not be compared to the 'H NMR spectra obtained from this
investigation. In future research these compounds will be isolated and characterized by NMR but
based on their structures
it
is anticipated that they would reside mainly in the hexanes layer as
well, since both are relatively large and non-polar molecules.
However, as
is the case for the
other compounds discussed,
it
is foreseeable that any compounds that are relatively non-polar
will reside
in both the hexanes and ethyl acetate
layers. As shown in Figure
10, compound 9
contains three terminal alkenes, which are anticipated to have chemical shifts of approximately 5
ppm.
Compound
11
contains three
internal alkenes, which are anticipated
to have chemical
shifts of approximately 6 ppm.
All fractions of the ethyl acetate layer exhibited bioactivity against both bacteria and are
marked in Scheme
1 (page 13) with an asterisk.
The bioassays were performed in triplicate and
the averages of the results are provided in Table
7. A table of all replications can be found ui
22
Appendix A-07.
Bacitracin was used
instead of penicillin
as
a control
for the background
bioactivity testing of the
ethyl acetate fractions due
to the unavailability of penicillin
at
that
particular time.
Substituting bacitracin for penicillin was of no consequence because
it was only
used as a control and not for synergistic testing with the plant extracts.
Table 7. Average zones of inhibition (mm) for ethyl acetate layer fractions
Bacillus subtilis
Staphylococcus aureus
Fraction
Blank
Bacitracin
(Ctrl)
lOjig
lOOfig
lOOOng
Blank
Bacitracin
(Ctrl)
lOHg
lOOfig
lOOOng
El
9.7
12.3
17.7
E2
9.3
12.6
18
7.7
E3
10
11
13.3
18.7
E4
10
12.7
15.3
17.3
10
11
E5
9.7
14.7
17
7.7
E6
9.7
8.7
11.3
18.7
8
Following the same procedure used
for the hexanes
layer,
the
fractions of the
ethyl
acetate layer were loaded onto discs preloaded with penicillin and ampicillin then subjected to
synergistic testing. The bioassays were duplicated and the averages of the results are provided in
Tables 8 and
9. All replications can be found
in Appendices A-08 and A-09. Some fractions
exhibited enhanced
bioactivity, some
fractions exhibited a decrease
in bioactivity, and some
fractions did not exhibit a change in bioactivity.
Fractions El, E3, E4, and E5 exhibited enhanced bioactivity against B.
subtilis when
combined with penicillin
or
ampicillin, and
fraction E2
exhibited enhanced bioactivity only
when combined with ampicillin. Fraction E4 exhibited enhanced bioactivity against
S. aureus
when combined with penicillin or ampicillin, and
fraction E3
exhibited enhanced bioactivity
when combined only with
penicillin.
Fraction E5
exhibited enhanced
bioactivity
against
S.
aureus when combined with penicillin, but unlike the other combinations the greater the amount
23
of plant extract added the
less enhanced the bioactivity exhibited.
Additive activity was not
observed because the zones of inhibition of the combinations were not equal to the sum of the
individual bioactivities of the controls and the fractions. But again,
if a surface area analysis is
performed
instead
of
a
linear
analysis
it
is
evident
that
the
enhancement
in
bioactivity
demonstrated was more than additive and congruent with both definitions of synergistic activity.
Fraction E6 exhibited a decrease in bioactivity against B.
subtilis when combined with
penicillin or ampicillin, and fraction E2 exhibited a decrease in bioactivity only when combined
with penicillin. Fraction E2 exhibited a decrease in bioactivity against S. aureus when combined
with penicillin or ampicillin, and fraction E5 exhibited a decrease only when combined with
ampicillin.
It was interesting to note that when tested against S. aureus all of these combinations
exhibited reverse synergy except the combination of fraction E6 with penicillin.
Despite that the
bioactivity
increased
the
greater
the amount
of plant
extract
added,
the
bioactivity of the
penicillin combined with 1000 j^g of fraction E6 was
still less than that of the penicillin control.
Fractions E2 and E6 did not exhibit an increase or decrease in bioactivity against S. aureus when
combined with penicillin or ampicillin, and fraction E3 exhibited an increase in bioactivity only
when combined with penicillin.
Table 8. Average zones of inhibition (mm) for synergistic testing
of the ethyl acetate fractions
Bacillus subtilis
Fraction
Blank
Pen.
(Ctrl)
Pen./
lOfig
Pen./
lOOng
Pen./
lOOOfig
Blank
Amp.
(Ctrl)
Amp./
10 US
Amp./
lOOfig
Amp./
lOOOjig
El
31.5
30
35.5
39.5
31.5
33.5
36.5
40
E2
32.5
31
31
30
29.5
29.5
32
35.5
E3
32
30
32.5
41
31
33.5
37
39.5
E4
30
29.5
31
35.5
32
29
30.5
33.5
E5
34.5
26.5
30
35.5
32.5
32.5
33.5
42
E6
33
31.5
32
32.5
33
32
31
31.5
24
Table 9. Average zones of inhibition (mm) for synergistic testing
of the ethyl acetate fractions
Staphylococcus aureus
Fraction
Blank
Pen.
(Ctrl)
Pen./
lOfig
Pen./
lOOjig
Pen./
lOOOng
Blank
Amp.
(Ctrl)
Amp./
10 fig
Amp./
lOOfig
Amp./
lOOOng
El
33.5
34
33.5
33
32
31
31.5
31.5
E2
34
33.5
33
32.5
32.5
30.5
32.5
31.5
E3
30
31.5
33.5
33.5
31.5
32
32
32
E4
29.5
30.5
30.5
24
32
31
29.5
42.5
E5
31
35.5
34
32.5
31.5
31
31
29.5
E6
33.5
33
33
33
31.5
31.5
32
31.5
25
~ CONCLUSION ~
Experiments by other researchers have investigated possible synergistic activity of plant-
derived compounds and
reports mention the demonstration of synergistic
activity but do not
provide the model of drug interaction on which the analysis was based.
'"^'^'^'^^ Because no clear
consensus
exists
pertaining
to
the
definition
of drug
synergism,
this
investigation merely
concludes
that the combination of a number of lemongrass {Cymbopogon flexuosus)
extracts
with the
antibiotics
penicillin and
ampicillin demonstrates some form of both enhanced and
reduced bioactivity. Whether or not synergistic activity was demonstrated by these samples
is
dependent on the reference model used for drug interaction. If drug synergism is defined as the
action
of one
drug
aiding
or enhancing
the
action
of another'^,
then drug synergism was
demonstrated in this investigation. If it is defined as the combined effect of drugs that exceeds
the sum
of
their
individual
effects '°,
then
drug
synergism was
not
demonstrated
in
this
investigation.
Critical
analysis of the results led
to the conclusion
that combining plant
extracts of
Cymbopogon flexiiousus with the known antibiotics penicillin and ampicillin did not result in
clear additive activity. This conclusion was based on the fact that when the zones of inhibition
were evaluated
linearly by measuring the diameters,
the
bioactivities of the combined plant
extracts and antibiotics were not equal to the sum of their individual bioactivities. However,
it is
interesting to note that when the zones of inhibition were evaluated by the areas and not by the
diameters, there was a clear enhancement in bioactivity that demonstrated more than additive
bioactivity and was congruent with both
definitions of synergistic
activity.
This method of
analysis will be addressed in a forthcoming paper.
26
~ EXPERIMENTAL ~
General.
All
solvents used were purchased from Aldrich Chemical Company'^ and were
distilled prior to use.
Final plant extracts were dried over Na2S04 prior to concentration. NMR
spectra were obtained on a JEOL ECX400 spectrometer in CDCI3 and internally referenced to
residual CHCI3 (7.24 ppm,
'H).
Plant material was collected and dried by Toni Thomas, an
extension agent of the University of the Virgin Islands co-op. extension service. All microbial
agents were purchased from Carolina Biological Supply Company.'^^ Blank discs (6 mm), agars,
and
nutrient
broth
were
purchased
from VWR
Scientific
Products. ^^
Antibiotic
discs
(penicillin/ampicillin, 6mm) were purchased from Ward's Natural Science Company.
Initial Extraction Process.
Weighed and milled plant material was placed in an Erlenmeyer
flask and soaked in MeOH for 24 h. The MeOH was decanted into a round-bottom flask, and the
process repeated twice more.
The combined extracts were concentrated in vacuo, weighed, and
set aside for subsequent extractions.
Liquid/Liquid Extraction Process.
Concentrated plant extract (330 mg) was dissolved in 80%
MeOH/H20 (100 mL), and a small amount of hexanes (10 mL) then transferred to a separator/
fimnel to be diluted in hexanes (total volume 100 mL) and partitioned.
The methanolic solution
was rinsed with hexanes
(2 x 40 mL) and the combined
extracts of the hexanes
layer were
concentrated in vacuo. The 80% MeOH/H20 solution was diluted to 30%) MeOH/H20 and rinsed
with EtOAc (3 x 40 mL).
The combined extracts of the EtOAc layer were dried over Na2S04
and concentrated in vacuo. The remaining MeOH/H20 layer was concentrated in vacuo as well.
27
TLC of the crude hexanes and EtOAc layers was performed on precoated silica gel 60 F254
plates and column cliromatography was carried out using 230-400 mesh sihca gel. The eluent
system used to separate the hexanes layer was 2:1 hexanes/EtOAc and the eluent system used to
separate the EtOAc layer was 1:1:1 acetone/pet ether/MeOH. A TLC of the crude hexanes layer
yielded two streaked spots with Rf values 0.37 and 0.71. A TLC of the crude ethyl acetate layer
yielded only one streaked spot with an Rf value of
0.70. All spots were observed by UV (254
nm) detection.
Acid/Base Extraction Process of Lemongrass.
Concentrated plant
extract (1.22
g) was
diluted in EtOAc (50 mL) and 0.1 M HCl (45 mL) and was then transferred into a separatory
funnel.
The layers were allowed to separate then partitioned.
The aqueous layer was extracted
with EtOAc
(2
x
40
mL).
The
combined
organic
layers were
dried
over Na2S04
and
concentrated
in vacuo.
To the resultant acidic layer was then added zinc dust (5
g) and the
mixture was allowed to stir at rt for approximately 3 h after which time the mixture was filtered
through a pad of Celite.
The mother liquor was then made basic (~ pH 9) using 25% NH4OH
and rinsed with EtOAc
(3 x 40 mL). The combined organic layers were dried over Na2S04 and
concentrated in vacuo. A TLC of the organic layer collected under acidic conditions was run in
4:1 hexanes/EtOAc and showed four spots observed by UV (254 nm) detection with the Rf
values
0,
0.17,
0.29,
and
0.88.
A TLC
of the
basic
organic
layer was
run
in
80:19:1
CHCl3/MeOH/NH40H and yielded only one spot with an Rfof 0.7.
Preparation of Agars,
Nutrient Agar- A IL bottle containing deionized water (500 mL) and
Nutrient Agar (11.5 g) was heated and stirred until the agar dissolved. The solution was allowed
28
to boil for
1 min, and was then autoclaved at 121 °C for 15 min.
After completing the autoclave
cycle the solution was allowed to cool, and the agar was poured into sterile Petri dishes that were
covered and left overnight. The following morning the Petri dishes were turned upside down and
refrigerated. Sabouraud Dextrose Agar- A
1 L bottle containing deionized water (500 mL) and
Sabouraud Dextrose agar (32.5 g) was heated and stiiTed until the agar dissolved.
The solution
was allowed to boil for
1 min, and was then autoclaved at 12 IT for 15 min. After completing
the autoclave cycle the solution was allowed to cool, and the agar was poured into sterile Petri
dishes that were covered and left overnight. The following morning the Petri dishes were turned
upside down and refiigerated.
Preparation of Nutrient Broth.
A
1 L bottle containing deionized water (500 mL) and
Nutrient Broth (4 g) was heated and stirred until the powder dissolved then allowed to boil for
1
min.
The mixture was autoclaved
at
121
°C
for
15 minutes.
After completing the autoclave
cycle the solution was allowed to cool to rt then refrigerated.
Preparation of Starter Plates.
A Petri dish filled with the appropriate agar for the microbial
agent to be plated (Nutrient Agar for Staphylococcus aureus, Sabouraud Dextrose for Bacillus
subtilis) was removed
from
the
refiigerator and
allowed
to warm
to
rt. An
ethanol/flame
sterilized inoculating hoop was used to remove a small amount of bacteria from a vial, which
was spread across one edge of the agar plate a few times.
The inoculating hoop was sterilized
once more by ethanol/flame and used to scrape approximately 2-3 lines from the starter colony in
another direction to start a new colony. The inoculating hoop was used to scrape 2-3 lines from
the second colony to start a third colony. The cultured Petri dish was allowed to incubate upside
29
down
at 37 °C
for approximately
18 h and stored
in the refrigerator upon removal from the
incubator.
Preparation of Starter Cultures.
Nutrient broth (5 mL) was transferred into a large autoclaved
vial and allowed to warm to
rt. An ethanol/flame sterilized inoculating hoop was used to scrape a
colony from the starter plate and transfer the microbial agent into the large vial containing the
nutrient broth.
The hoop was rotated numerous times to ensure that the tip of the hoop came in
contact with the bottom of the vial.
The inoculated broth was incubated at 37 °C for 4-6 h and
gently agitated approximately every 30 min.
Process
of Loading
Discs.
The
concentrated
plant
extract
(20 mg) was
diluted with
the
appropriate amount of solvent (2 mL) to make a 10 mg/mL solution referred to as Solution A.
One half millihter of Solution A was transferred into a vial and diluted to 10 mL with the same
solvent to yield a 500 [ig/mL solution referred to as Solution B. The discs were placed on a
watch glass and loaded one drop at a time while directing a stream of air at the discs for drying
purposes.
For discs containing
1
p,g of the plant extract the discs were loaded with 20
|a.L of
Solution B.
For discs containing 100 [ig of the plant extract the discs were loaded with 10
|j,L of
Solution A.
For discs containing
1 mg of the plant extract the discs were loaded with 100
(j,L of
Solution A.
After loading, the discs were placed in a vacuum dessicator and concentrated in
vacuo for a minimum of 20 min.
Preparing Culture Dishes.
Petri dishes, filled with the appropriate agar for the microbial agent
to be plated, were removed from the refrigerator and allowed to warm to rt.
Using an autoclaved
30
Eppendorf pipet, 100 [iL of the starter culture was transferred into the center of the Petri dish.
An ethanol/flame sterilized spreader was used to disperse the bacteria evenly across the agar and
the bacterial solution allowed to dry for no more than 5 min.
Using autoclaved tweezers, the
discs were applied to the agar a considerable distance from each other (~ 2.5 cm) and from the
sides of the Petri dish (~1 cm).
The dish was turned upside down and incubated at 37 °C for
approximately
1 8 h.
Measurement of the Zones of Inhibition. The cultures were removed from the 37 °C incubator
after approximately
18
h.
Using a
ruler,
the zone of inhibition
(in mm.)
for each disc was
determined by measuring the diameter of the area in which bacterial growth was inhibited.
Synergistic Testing Process.
The discs to be used for synergistic testing were prepared in the
same manner as outlined in the experimental with the exception that the extracts were loaded
onto discs preloaded with penicillin (10 lU) or ampicillin (10 ^g). The process of synergistic
testing was identical to the process used for background bioactivity testing.
Storage of Microbes. All microbes were stored in a 37 °C incubator.
31
~ FUTURE RESEARCH ~
This investigation was initiated as a pilot study for many future investigations.
The next
investigation will be the synergistic testing of the pure bioactive components of the Cymbopogon
flexnosus taxa used in this investigation.
Through an extensive literature search,
all compounds
within
the
plant
that
are known
to
exhibit
bioactivity
will be purchased and
subjected
to
synergistic testing with penicillin, ampicillin, and possibly other antibiotics such as streptomycin.
All other compounds will be isolated and characterized, and those that exhibit bioactivity will be
subjected to synergistic testing with known antibiotics as well.
Another type of synergistic testing to be investigated
is the possible synergy between
compounds within the plant.
Plants usually present defenses as a suite of compounds, not as
individual
ones."^
It
is thought
that minor constituents found
in low percentages may act as
synergists,
enhancing
the
effectiveness
of
the
major
constituents
through
a
variety
of
mechanisms. ''
The
results
of
an
investigation
of
possible
synergistic
activity
between
compounds within the plant can then be applied to further synergistic testing of plant extracts
and antibiotics. After thoroughly investigating possible synergistic activity between lemongrass
plant
extracts and
antibiotics,
possible
synergistic
activity between
the
plant
extracts
and
fungicides can be investigated. Lemongrass has been found to exhibit antifungal
activity, with
citral as one of the fungicidal constituents.
'"
These investigations will be performed on other medicinal herbs from the United States
Virgin
Islands,
particularly worrywine
{Stachytarpheta jamaicensis),
beggar's
tick
{Bidens
bipinnata), inflammation bush {Verbesina alata), and noni {Morinda citrifolia).
32
~ REFERENCES ~
1
.
Summer, J. The Natural History of Medicinal Plants , Timber Press, Inc. Portland, Oregon,
2000.
2.
Levetin, E.; McMahon, K. Plants and Society . McGraw-Hill, 2003.
3.
Natural Products as Medicinally Useful Agents.
http://www.people.vcu.edu/~asneden/MEDC%203105%20Intro.htm Accessed on 9
January 2004.
4.
Quinine. http://www.eDcvclopedia.coni/html/ql/qumine.asp Accessed on 28 January
2004.
5.
Coombes, J. D., Ph.D, ed. New Drugs from Natural Sources . IBC Technical Services Ltd.:
Great Britain, 1992.
6.
Rouhi, Maureen A. Chemical & Engineering News 2003, 81, (41), 93-103.
7.
Newman, D.J.; Cragg, G.M.; Snader. K.M. J. Nat. Prod. 2003, 66, 1022-1037.
8.
CDC report: Background on Antibiotic Resistance.
http://www.cdc.gov/drugresistance/community/ Accessed on 27 Aug. 2003.
9.
Wang, Y; Zuo, J.; Wientjes, M. "Quantiation of Interactive Effects between Anticancer
Agents Using SAS® Macros and SAS/Graph®"
10. Cappelletty, D. M; Rybak, M.J. J. Antimicrobial Agents and Chemotherapy 1996, 40, (3),
677-683.
11. Wellman, P.J.; Maher, T.
J. Int. J. Relat. Metab. Disord. 1999, 23, (7), 723-732.
12. Hummelbrunner, L.; Isman, M. J. Agric. Food Chem. 2001, 49, 715-720.
13. Guz, N.R.; Stermitz, F.R.; Johnson, J.B.; Beeson, T.D.; Willen, S.; Hstang, J-F.; Lewis, K.
J.
Med Chem. 2001, 44, (2), 261-268.
14. Lemongrass. http://www.hort.purdue.edu/newcrop/med-
aro/factsheets/LEMQNGRASS.html Accessed on 2 February 2004.
15. Chao, S.C; Young, D.G.; Oberg, C. J. Journal ofEssential Oil Research 2000, 12, (5), 639-
649.
16. Lawless, J. Illustrated Encyclopedia of Essential Oils: The Complete Guide to the Use of Oils
in Aromatherapy and Herbalism. Element Books, Rockport, MA, 1995, 56-67.
33
17. Qamar, S.; Hanif, M.; Chaudhary, F.M.. Pak. J.Sci. hid. 1992, 35, (6), 246-249.
18. Penicillin. http://ww"w.sbodor.ore/niaster/biomed/pharmaco/peDicilliD/penapp.html
Accessed on September 2003.
19. Ampicillin.
http://www.medicinenet.coni/ampicilliii/article.htm Accessed on 9 February.
2004.
20. Bacillus subtilis. http://www.micron.ac.uk/Organisms/BaciIlus/Bsubtilis.htmJ Accessed
on 27 January 2004.
21. Singleton, P.; and D. Sainsbury. Introduction to Bacteria: for students in the biological
sciences. John Wiley and Sons, Binghamton, NY, 1981, 150-151.
22. VISA/VRSA Fact Sheet, http://www.cdc.gov/ncidod/hip/aresist/visa.htm Accessed on 2
February 2004.
23. Baldry, P. The Battle Against Bacteria . Cambridge University Press, 1976, 33.
24. MicroBioNet. Bacteriology,
http://www.sciencenet.com.au/gramnegativebacteria.htm
Accessed on 27 January 2004
25. Procaryotic Cell Structure and Function.
http://www.uta.edu/biologv/badon/classnotes/3444/Lecture4.pdf Accessed on
1
7
February 2004.
26. Cimanga, K.; Kambu, K.; Tona, L.; Apers, S.; De Bruyne, T.; Hermans, N.; Totte, J.; Pieters,
L.; Vlietinck. A. Journal ofEthnopharmacology 2002, 79, 213-220.
27. Alves, A. C; Prista, L. N.; Ferreira, A. M. Garcia de Orta 1962, 10, (1), 103-109.
28. Mattocks, A. R. Chemistry and Toxicology of Pyrrolizidine Alkaloids. London: Academic
Press, Inc., 1986.
29. Nath, S.; Sarma, K.; Vajezikova,
I.; Leclercq, P. Biochemical Systematics and Ecology 2002,
30,151-162.
30. Onawunmi, G.; Yisak, W.; Ogunlana, E. Journal ofEthnopharmacology 1984, 12, 279-286.
31. Begum, J.; Yusuf, M.; Chodhury, J.; Wahab, M. Bangladesh J Sci, hid Res. 1993, 28, (4),
26-34.
34
32. Schaneberg, B.; Khan.
I. J. Agric. Food Chem. 2002, 50, 1345-1349.
33. Taskinen, J.; Mathela, D.; Mathela, C. Journal of Chromatography 1983, 262, 364-366.
34. Paranagama, P.; Abeysekera, K.; Abeyeickrama, K.; Nugaliyadde, L. Letters in Applied
Microbiology 2003, 37, (1), 86.
35. Khalaf, J.; Datta, A. J. Org Chem. 2003, 69, 387-390.
36. Aldrich Chemical Company.
P.O. Box 355. Milwaukee, WI
53201.
37. Carolina Biological Supply Company.
P.O. Box 6010.
Burlington, NC 27216-6010.
38. VWR International.
P.O. Box 5015.
Bristol, CT 06011-8960.
39. Ward's Natural Science.
P.O. Box 92912.
Rochester, N^'
14692-9012.
40. Microbiological Techniques.
httD://web.ukonliDe.co.uk/webwise/sDiDDeret/microbes/techl.htm
Accessed on 14
February 15, 2004.
35
~ Appendix A -
Tables of Replications
A-01
Replications of Bioactivity Testing of Crude Lemongrass Extract
A-02
Replications of Bioactivity Testing of Crude Liquid/Liquid Partition Layers of
Lemongrass against Bacillus subtilis
A-03
Replications of Bioactivity Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Staphylococcus aureus
A-04
Replications of Synergistic Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Bacillus subtilis
A-05
Replications of Synergistic Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Staphylococcus aureus
A-06
Replications of Bioactivity Testing of Hexanes Fractions against Staphylococcus aureus
A-07
Replications of Synergistic Testing of Hexanes Fractions and Penicillin
against Bacillus subtilis
A-08
Replications of Synergistic Testing of Hexanes Fractions and Ampicillin
against Bacillus subtilis
A-09
Replications of Synergistic Testing of Hexanes Fractions and Penicillin
against Staphylococcus aureus
A-10
Replications of Synergistic Testing of Hexanes Fractions and Ampicillin
against Staphylococcus aureus
A-1 1
Replications of Bioactivity Testing of Ethyl Acetate Fractions against Bacillus subtilis
A-12
Replications of Bioactivity Testing of Ethyl Acetate Fractions
against Staphylococcus aureus
A-1 3
Replications of Synergistic Testing of Ethyl Acetate Fractions and Penicillin
against Bacillus subtilis
A-14
Replications of Synergistic Testing of Ethyl Acetate Fractions and Ampicillin
against Bacillus subtilis
A-1 5
Replications of Synergistic Testing of Ethyl Acetate Fractions and
Penicillin against Staphylococcus aureus
A-1 6
Replication of Synergistic Testing of Ethyl Acetate Fractions and Ampicillin
against Staphylococcus aureus
36
~ APPENDIX A ~
*The results of the replications below are shown in the following manner:
Replication
1, Replication 2, RepUcation 3
Al. Replications of Bioactivity Testing of Crude Lemongrass Extract
Bacillus subtilis
Staphylococcus aureus
Blank
Penicillin
(Ctrl)
lo^g
lOOfig
lOOOng
Blank
Penicillin
(Ctrl)
lO^ig
lOOjig
lOOOng
0,0
35,27.5
0,0
1
8,0
8.5,8.5
0,0
31.5,34
0,0
8,9
11, 13
A2. Replications of Bioactivity Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Penicillin (ctrl)
10 US
100 ng
1000 ^g
Hexanes
0,0,0
24, 27, 27
0,0,0
8.5,9,8.5
9, 11, 10
Ethyl acetate
0,0,0
34, 30, 35
0,0,0
6.5,7,6.5
13, 11, 11
Methanol/H20
0,0,0
32.5,32.5,36
0, 0,
0, 0,
0,0,0
A3. Replications of Bioactivity Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Penicillin (ctrl)
10 ^g
100 fig
1000 fig
Hexanes
0,0,0
33.5,33.5,33.5
0,0,0
0,0,0
0,0,0
Ethyl acetate
0,0,0
34,35,35
0,0,0
0,0,0
12, 11.5, 13
Methanol/HiO
0,0,0
27,33,37
0,0,0
0,0,0
0,0,0
37
A4. Replications of Synergistic Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Penicillin (ctrl)
Pen./
100 ng
Pen./
1000 ^g
Hexanes
0,0,0
33,35.36
33,35,34
30, 29, 30
Ethvl acetate
0,0,0
27,35,38
27, 32, 32
27, 23, 29
A5. Replications of Synergistic Testing of Crude Liquid/Liquid Partition Layers
of Lemongrass against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Penicillin (ctrl)
Pen./
100 ng
Pen./
1000 ng
Hexanes
0,0,0
34, 34, 35
32, 34, 32
32.5,32,31
Ethvl acetate
31,34,33
31,32,30
28,28,29
A6. Replications of Bioactivity Testing of Hexanes Fractions against Staphylococcus aureus
Staphylococcus aureus
\
Fraction
Blank
Penicillin (ctrl)
10 Jig
100 Jig
1000 fig
HI
0,0
36,35
0,0
0,0
0,0
H2
0,0
35,36
0,0
9, 11
9,8
H3
0,0
35,37
0,0
10, 14
10, 15
H4
0,0
36,38
0,0
10, 12
10,N/A
H5
0,0
35,34
0,0
10, 12
13, 13
H6
0,0
36,35
0,0
10, 11
18,16
H7
0,0
34,35
0,0
9,9
13, 13
H8
0,0
38,38
0,0
8,7
11, 12
H9
0,0
36,37
0,0
7,9
10,7
A7. Replications of Synergistic Testing of Hexanes Fractions and
Penicillin against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Pen. (Ctrl)
Pen./
lOfig
Pen./
lOOfig
Pen./
lOOO^g
HI
0,0,0
32, 29, 32
34,31,25
35,34,30
38,37,34
H2
0,0,0
33,33,33
31,28,31
36,35,36
39,37,40
H3
0,0,0
32,30,27
35,32,28
37,35,33
39,41,40
38
A8. Replications of Synergistic Testing of Hexanes Fractions and
Ampicillin against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Amp. (Ctrl)
Amp./
10 US
Amp./
lOOng
Amp./
lOOOfig
HI
0,0,0
35,35,36
30, 30, N/A
31, 34, N/A
43,38,38
H2
0,0,0
31,33,32
31,32,30
33,35,34
40, 39, 35
H3
0,0,0
32,32,32
31,32,32
35,36,35
39, 40, 39
A9. Replications of Synergistic Testing of Hexanes Fractions and
Penicillin against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Pen. (Ctrl)
Pen./
lOng
Pen./
100^g
Pen./
lOOOHg
HI
0,0,0
34, 34, 34
34, 35, 34
34, 35, 33
34,35,33
H2
0,0,0
33, 32, 34
33,32,33
33,31,33
32,31,33
H3
0,0,0
31,32,34
32,31,32
33,31,32
35,33,33
AlO. Replications of Synergistic Testing of Hexanes Fractions and
Ampicillin against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Amp. (Ctrl)
Amp./
10 ng
Amp./
lOOjig
Amp./
lOOO^g
HI
0,0,0
31,32,32
31.32,32
31,32,32
31,31,31
H2
0,0,0
30, 30, 30
30, 30, 30
30, 30, 30
30, 30, 30
H3
0,0,0
35,32,32
31,32,32
29, 29, 30
29, 30, 30
All. Replications of Bioactivity Testing of Ethyl Acetate Fractions against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Bacitracin (ctrl)
lOfig
lOOng
lOOOjig
El
0,0,0
10, 10,9
0,0,0
0,0,0
12, 12, 13
E2
0,0,0
9, 10,9
0,0,0
0,0,0
12, 12, 14
E3
0,0,0
10,10,10
0,0,0
11, 12,10
12,15,13
E4
0,0,0
10, 10, 10
0,0,0
12, 14, 12
15,16,15
E5
0,0,0
10, 10,9
0,0,0
0,0,0
19, 15, 10
E6
0,0,0
10,9, 10
0,0,0
9,9,8
11, 13, 10
39
A12. Replications of Bioactivity Testing of Ethyl Acetate Fractions
against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Bacitracin (ctrl)
lOfig
loo^g
lOOO^g
El
0,0,0
18, 17, 18
0,0,0
0,0,0
0,0,0
E2
0,0,0
18, 18, 18
0,0,0
0,0,0
8,7,8
E3
0,0,0
19, 18, 19
0,0,0
0,0,0
0,0,0
E4
0,0,0
17, 18,17
0,0,0
10. 10, 10
11, 12, 10
E5
0,0,0
17, 17, 17
0,0,0
0,0,0
7,8,8
E6
0,0,0
19, 18, 19
0,0,0
0,0,0
8,8,8
A13. Replications of Synergistic Testing of Ethyl Acetate Fractions and
Penicillin against Bacillus subtilis
Bacillus subtilis
Fraction
Blank
Pen. (Ctrl)
Pen./
lO^ig
Pen./
lOOjig
Pen./
lOOOfig
El
0,0
32,31
29,31
37,34
39,40
E2
0,0
33,32
31,31
31,31
30,30
E3
0,0
33,31
30,29
32,32
35,36
E4
0,0
30, N/A
31,28
32,30
36,35
E5
0,0
34,35
28,25
30,30
36,35
E6
0,0
33,32
34,31
33,34
42,42
A14. Replications of Synergistic Testing of Ethyl Acetate Fractions and
Ampicillin against Bacillus subtilis
Bacillus subtilis
\
Fraction
Blank
Amp. (ctrl)
Amp./
10 ^g
Amp./
lOOfig
Amp./
1000^g
El
0,0
31,32
34,33
36,37
40,40
E2
0,0
30,29
30, 29,
32,32
35,36
E3
0,0
31,31
32,35
37,37
40,39
E4
0,0
34,30
29,29
29,32
32,35
E5
0,0
33,32
34,31
33,34
42,42
E6
0,0
33,33
32,32
31,31
32,31
40
A15. Replications of Synergistic Testing of Ethyl Acetate Fractions and
Penicillin against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Pen. (Ctrl)
Pen./
lOjig
Pen./
lOOng
Pen./
lOOOfig
El
0,0
33,34
34,34
33,34
33, N/A
E2
0,0
34,34
33,34
34,32
33,32
E3
0,0
31,29
32,31
35,32
35,32
E4
0,0
30,29
30,31
31,30
22,26
E5
0,0
30,32
36.35
35,33
33,32
E6
0,0
33,34
33,33
33,33
33,33
A16. Replication of Synergistic Testing of Ethyl Acetate Fractions and
Ampicillin against Staphylococcus aureus
Staphylococcus aureus
Fraction
Blank
Amp. (Ctrl)
Amp./
10 fig
Amp./
lOOfig
Amp./
lOOOfig
El
0,0
32,32
31,31
31,32,
31,32
E2
0,0
32,33
30,31
33,32
31,32
E3
0,0
32,31
32,32
32,32
32,32
E4
0,0
32,32
31,31
30,29
24,25
E5
0,0
32,31
32,30
32,30
28,31
E6
0,0
31,32
31,32
32,32
31,32
41
~ Appendix B ~
Lemongrass 'H NMR Spectra
Page
'H NMR spectrum of A/B extraction organic layer under acidic conditions
44
'H NMR spectrum of A/B extraction organic layer under basic conditions
45
H NMR spectrum of Liquid/Liquid Partition crude hexanes layer
46
"H NMR spectrum of Liquid/Liquid Partition crude ethyl acetate layer
47
H NMR spectrum of Liquid/Liquid Partition crude methanol layer
48
H NMR spectrum of fraction HI from hexanes layer column A
49
H NMR spectrum of fraction H2 from hexanes layer column A
50
'H NMR spectrum of fraction H3 from hexanes layer column A
51
'H NMR spectrum of fraction H4 from hexanes layer column A
52
H NMR spectrum of fraction H5 from hexanes layer column A
53
H NMR spectrum of fraction H6 from hexanes layer column A-
54
'H NMR spectrum of fraction H7 from hexanes layer column A
55
'H NMR spectrum of fraction H8 from hexanes layer column A
56
H NMR spectrum of fraction H9 from hexanes layer column A
57
'H NMR spectrum of fraction HI from hexanes layer column B
58
'H NMR spectrum of fraction H2 from hexanes layer column B
59
H NMR spectrum of fraction H3 from hexanes layer column B
60
H NMR spectrum effraction H4 from hexanes layer column B
61
H NMR spectrum of fraction H5 from hexanes layer column B
62
H NMR spectrum of fraction H6 from hexanes layer column B
63
42
B-21
'H NMR spectrum of fraction H7 from hexanes layer column B
64
B-22
'H NMR spectrum of fraction H8 from hexanes layer column B
65
B-23
'H NMR spectrum of fraction El from ethyl acetate layer column
66
B-24
'H NMR spectrum of fraction E2 from ethyl acetate layer column
67
B-25
'H NMR spectrum of fraction E3 from ethyl acetate layer column
68
B-26
'H NMR spectrum of fraction E4 from ethyl acetate layer column
69
B-27
'H NMR spectrum of fraction E5 from ethyl acetate layer column
70
B-28
'H NMR spectrum of fraction E6 from ethyl acetate layer column
71
B-29
H NMR spectrum of citral (mixture of cis and trans isomers)
72
B-30
'H NMR spectrum of linalool
73
B-31
'H NMR spectrum of P-caryophyllene oxide
74
43
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