A SEARCH FOR MULTI-DRUG RESISTANCE PUMP INHIBITOR
MOLECULES BY ISOLATION OF NATURAL PRODUCTS
Approved by:
______Edward R. Biehl, Professor
______John A. Maguire, Professor
______David Y. Son, Professor
______Pia D. Vogel, Professor
A SEARCH FOR MULTI-DRUG RESISTANCE PUMP INHIBITOR
MOLECULES BY ISOLATION OF NATURAL PRODUCTS
A Thesis Presented to the Graduate Faculty of
Dedman College
Southern Methodist University
In
Partial Fulfillment of the Requirements
For the degree of
Master of Science
With a
Major in Chemistry
By
Alan Wilfred Humason
(B.S., Chemistry, University of Massachusetts at Amherst)
May 14, 2005
Humason, Alan B.S., University of Massachusetts at Amherst, 1979
A Search for Multi-Drug Resistance Pump Inhibitor Molecules By Isolation of Natural Products
Advisor: Professor Edward R. Biehl
Master of Science conferred May 14, 2005
Thesis completed April 22, 2005
A search for multi-drug resistant pump inhibitory molecules was conducted in several plants that have clinically, or in traditional medicine, demonstrated useful activity against bacteria. The investigation was conducted by extracting the plants with organic solvents, and fractionating the resultant extracts chromatographically. These fractions were then subjected to testing against Staphylococcus aureus, in conjunction with a sub-inhibitory dose of the antibiotic berberine.
Extracts exhibiting inhibitory activity were further fractionated using vacuum liquid chromatography, flash liquid chromatography and conventional and preparative thin layer chromatography, attempting to isolate a single molecule which exhibits multi- drug resistance pump inhibition.
The plants that were investigated, Eriogonum brevicaule Nuttall, Hydrastis canadensis, Thuja occidentalis, Rhus trilobata, Pouteria pallida, and Gunnera macrophylla, yielded over two hundred fractions, including several active ones. The utility of each of the chromatographic techniques was demonstrated and critiqued.
iii
TABLE OF CONTENTS
LIST OF FIGURES...... v
LIST OF TABLES ...... vi
ACKNOWLEDGEMENTS ...... vii
Chapter
I. INTRODUCTION...... 1
II. EXPERIMENTAL SECTION...... 7
III. EXTRACTION PARAMETERS FOR SPECIFIC SAMPLES ...... 13
Eriogonum brevicaule Nuttall ...... 13
Hydrastis canadensis...... 24
Pouteria pallida – N014795...... 27
Thuja occidentalis – N102279/ N102249...... 30
Rhus trilobata – N102791 ...... 32
Gunnera macrophylla – N014045...... 35
IV. MDRP ACTIVITY SUMMARY ...... 37
V. CONCLUSION ...... 45
Appendices
A. MOLECULAR STRUCTURE OF CERTAIN ANTIBIOTICS ...... 48
B. EXTRACTION LOG...... 49
C. MDRP ACTIVITY TESTING RESULTS...... 54
D. SPECTROSCOPIC ANALYSIS BY DR. RAMADAS SATHUNURU...... 67
REFERENCES...... 73
iv
LIST OF FIGURES
Figures
1. Molecular Structures – Reserpine ...... 4
2. Molecular Structure – 5”-Methoxy hydrocarpin-D...... 4
3. Molecular Structure – Berberine ...... 5
4. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid...... 43
5. Molecular Structure of Certain Antibiotics ...... 48
6. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid...... 67
7. Molecular Structure – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid acetate...... 68
8. HRMS – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid...... 70
9. 1H NMR – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid...... 71
10. 1H NMR – 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid acetate ...... 72
v
LIST OF TABLES
Table
1. Eriogonum brevicaule Nuttall Hillside Fractionation Parameters ...... 18
2. Eriogonum brevicaule Nuttall FxCP Fractionation Parameters...... 19
3. Eriogonum brevicaule Nuttall Leaves Fractionation Parameters...... 19
4. Eriogonum brevicaule Nuttall Roots Fractionation Parameters...... 20
5. Eriogonum brevicaule Nuttall Re-extraction Parameters...... 21
6. Flash Chromatography Parameters for AH73/AH103/AH127 ...... 23
7. Flash Chromatography Parameters for Pouteria pallida...... 28
8. Flash Chromatography Parameters for Thuja occidentalis ...... 31
9. Flash Chromatography Parameters for Rhus trilobata AH250 ...... 34
vi
ACKNOWLEDGMENTS
I would like to thank the many scientists who assisted me with this paper, both with information and with their efforts.
Dr. Frank Stermitz, who provided flora samples and technical advice.
Dr. Kim Lewis, whose laboratories at Tuft’s University and Northeastern
University performed the biological activity testing, and who also assisted with technical advice.
Dr. George Tegos, and Mr. Anthony Ball, who did the activity testing, under
Dr. Lewis.
Dr. Edward Biehl, who was my thesis advisor and mentor.
Dr. Pia Vogel, who reviewed and corrected the biological references in this thesis, and the thesis itself, thus completing her review twice.
Dr. Ramadas Sathunuru, who gave me invaluable guidance and assistance in the laboratory, as well as investing his own time in this research. It is his work that appears in Appendix D.
I must also express my thanks and affection to my wife, Melissa McNamara
Humason, and my children, Alana Rose Humason and Aidan Bartholomew Humason, for their support and their acceptance of my absences while completing this work.
vii
Chapter I
INTRODUCTION
To describe the search for a multi-drug resistance pump inhibitor, one must first define the terms. We can actually build this definition one term at a time.
In the counter-action of bacteria, the traditional medical approach is to use antibiotics, such as penicillin1,2, tetracycline3, erythromycin4, and the “last resort”5 antibiotic vancomycin.6 These are the drugs.
The need has always existed for the development of new antibiotics, because the old ones become ineffective as bacteria become resistant to them.7-10 This is the drug resistance.
One method used by bacteria to counteract drug therapy is to develop a mechanism to eject the antibiotics from each cell. This mechanism takes the form of a complex protein that spans the cell wall. This protein is programmed to identify an antibiotic and systematically pump it outside the cell wall. This is the drug resistance pump. As new antibiotics are developed, the bacterial drug resistance pumps succeed in ejecting the new drug, which may be chemically very different from old ones. The structural diversity of current antibiotics is shown in Appendix A. This ability to eject many different antibiotics is what constitutes the multi-drug resistance pump
(MDRP.)11
1
The anthropogenic approach to overcoming the MDRP is not useful for flora.
Plants have the ability to produce their own antibiotics. For example, Hydrastis canadensis, known as goldenseal, is a source of the naturally occurring antibiotic berberine. The plant does not, however, have the ability to create new antibiotics as the bacteria reject the old ones. Therefore, flora takes a different approach. This defense mechanism is to create a molecule, dissimilar to the antibiotic, which can disrupt the pump mechanism. These molecules will enter the multi-drug resistance pump and bind, thereby blocking the bacteria’s antibiotic defense. Then, the original antibiotics become efficacious again. These compounds are the multi-drug resistance pump inhibitors.12
The goal of this study is to identify one or more compounds that demonstrate multi-drug resistance pump inhibition. Plants that have shown useful activity against bacteria may employ MDRP inhibitors, although they may use other defense mechanisms. These plants are the focus of this study. The manner of the search is outlined as follows:
1) Obtain samples of those plants that have clinically, or in traditional medicine,
been used antibacterially.
2) Isolate the smaller molecular weight substituents from these plants by a series of
solvent extractions.
3) Test these extracts for multi-drug resistance pump inhibition.
4) Further fractionate the active fractions, ultimately into individual compounds.
5) Once again, test the individual compounds for pump inhibition.
2
6) Identify the individual compounds by nuclear magnetic resonance and mass
spectroscopy.13
7) Ultimately, synthesize the suspected multi-drug resistance pump inhibitor
molecule and test the synthetic compound. This will conclusively identify the
multi-drug resistance pump inhibitor.
Multi-drug Resistance Pump Inhibition Testing.5,12 The testing for MDRP inhibition activity was conducted at Tufts University, Sommerville, Massachusetts, and
Northeastern University, Boston, Massachusetts. At both institutions, Dr. Kim Lewis was the lead investigator, with Dr. George Tegos participating in the testing. Similar testing was conducted on the crude extracts and the refined fractions. In each case, a series of parallel experiments were conducted. The antibiotic used for this study was berberine, and the bacterium was Staphylococcus aureus (S. aureus). Berberine was administered at 30 micrograms per liter, approximately one sixth the minimum inhibitory concentration (MIC) of 120-240 micrograms per liter. The parallel tests included:
Cells. This sample contains the bacterium S. aureus in the nutritive medium
Mueller-Hinton Broth (MHB). S. aureus has been found to employ the translocation protein NorA as a multi-drug resistance pump.5 Naturally occurring compounds which have demonstrated inhibitory activity include reserpine12,14 and 5”-methoxy hydrocarpin-D12 (Figures 1,2.) The bacteria are expected to grow unchecked in this sample, and provide an upper limit for total growth. Growth was measured for this, and all other tests, using a microtiter plate reader (Biorad) by absorption at 600nm.12
3
H MeO N H H O H MeO OMe O O OMe OMe
OMe
Figure 1. Molecular Structure - Reserpine
O OH HO O O OMe
OH OH O OMe
Figure 2. Molecular Structure – 5”-Methoxy hydrocarpin-D
Berberine. This sample contains the bacterium and the antibiotic berberine
(Figure 3). The bacteria are also expected to grow unchecked in this sample, because the antibiotic was administered below the MIC for S. aureus. The optimum result would be growth identical to the “cells” sample. If growth is retarded, then the original
MIC assumption is called into question.
4
O O
N CH O 3 + OMe
Figure 3. Molecular Structure - Berberine
Extract. This sample contains an aliquot of the extract obtained from the plant under investigation and the bacterium, but no antibiotic. In this test, the desired result once again is to see unchecked growth of the S. aureus. If the bacteria are stunted by the extract alone, then the extract itself is poisonous to the bacteria. It means that the extract may also prove poisonous to a human host, if used medicinally. It will, in any event, mask any MDRP inhibition activity. An extract that retards cell growth at this step is said to demonstrate “direct activity”, and is not studied further. Note that this does not necessarily mean that the extract does not contain a MDRP inhibitor, as this compound may be present along with a different compound which is the bacterium killer.
Extract, Berberine and Cells. In this set of experiments, the S. aureus bacterium, berberine antibiotic and plant extract are all combined. A positive result would be a reduction in bacteria growth, which indicates that a MDRP inhibitor may be present.
The extract is introduced at four different concentrations, to establish the minimum inhibitory concentration. The most frequently used concentrations were
5
500 µg/mL, 250 µg/mL, 125 µg/mL and 62.5 µg/mL, or 50 µg/mL, 25 µg/mL, 12.5
µg/mL and 6.25 µg/mL. Often it is observed that growth is completely stopped in the more concentrated samples and partially stopped or unstopped in the more dilute samples.
MHB. The last experiment is a sample of unmodified Mueller-Hinton Broth nutritive medium. This is a control experiment that will show no growth, and define a zero point for the other tests. Growth in this sample will indicate that the plate is contaminated, and no useful results may be obtained.
Bioactivity data presented in this study will provide growth values for each of these tests, measured in terms of the opaqueness of each sample. The MHB sample is expected to be least opaque, and may be considered a zero point. The Cells sample is expected to be most opaque.
6
Chapter II
EXPERIMENTAL SECTION
Several extraction techniques were employed, for the raw plants and plant extracts which were fractionated. An overview of each technique follows.
Raw Plants. Raw plants were ground and extracted as described by Stermitz, et.al.13,15 The samples were homogenized in a stainless steel Waring Commercial
Blender. In the cases where the plants were separated into leaf, root and flower, the fragments were extracted separately.
The homogenate was then weighed, placed in a 4L Erlenmeyer flask and one liter of hexane was added. The mixture was swirled, and left to stand for a minimum of
24 hours. The mixture was then gravity filtered to isolate the hexane extract.13 The extract was concentrated on a rotary evaporator (Yamato RE 200) to remove the hexane, and the resultant material was set aside for further testing.
The filter cake from this procedure was returned to the 4L flask, and one liter of ethyl acetate was introduced. This mixture was also swirled and left to stand for 24 hours. The ethyl acetate extract was similarly isolated.
The twice-extracted filter cake was then extracted with methanol for 24 hours, the methanol extract was isolated, and the filter cake was discarded.
7
As previously stated, Dr. Stermitz provided the choice of extraction solvents.
Control experiments using solvents but no plant extracts were not conducted at
Southern Methodist University. Evaluations of some of the extraction solvents have been conducted by other researchers.10,15
Raw Plant Extracts. The extracts generated by the raw plant procedure outlined above were further fractionated by vacuum liquid chromatography.16,17 The detailed procedure is as follows:
A 350mL sintered glass buchner funnel was loaded with approximately 100g silica gel (Silicycle, Catalog # R12030B.) The silica gel was used as received, without activation by heat or deactivation with water. The buchner funnel was placed on a 2L filter flask, and a vacuum was drawn using a water aspirator. The silica filter bed was then compacted, using a large, black rubber stopper, to make a solid bed. This filter bed was then washed with two 100ml portions of hexane, and the filtrate was discarded.
After setting aside an aliquot of approximately 20mg for MDRP inhibition testing, the raw plant extract was then dissolved, or suspended, in a minimum amount of acetone. Silica gel was added to this mixture until the mixture became free flowing and visually dry. This silica suspension was gently poured onto the top of the prepared silica bed, while the vacuum was being applied. The silica suspension was compacted with the rubber stopper, and a clean filter paper was placed on top of the filter bed to prevent disturbance by the introduction of solvent.
This filter bed was then eluted with several 50mL portions of hexane until the eluent ran clear. This was typically approximately 300mL of solvent. After the last
8
portion was added, the apparatus was allowed to run under vacuum for 5 minutes, to isolate the majority of the solvent. The color of the eluent was noted, and the portions were combined, concentrated by rotary evaporation, and set aside for MDRP inhibition testing.
The filter bed was then eluted with a mixture of 1:1 hexane: ethyl acetate, adding 50mL portions until the eluent ran clear. Again, the color was noted, portions combined, and the concentrated extract was set aside for testing.
By this same procedure, the filter bed was extracted with pure ethyl acetate, 1:1 ethyl acetate: methanol, and pure methanol. Each time, elution was continued until the eluent ran clear. This yielded a total of five fractionated extracts, in addition to the original unfractionated extract.
Thus, with the fractionation of original hexane, ethyl acetate and methanol extracts, there resulted a total of eighteen extracts for testing. However, upon concentration, some of the fractions yielded no material, or too little to be submitted for testing.
Active Extracts. Extracts that demonstrated biological activity, and also some extracts which were supplied by Northeastern University and believed to be biologically active, were subjected to silica gel column chromatography and preparative thin layer chromatography.
Silica Gel Column Chromatography. A liquid chromatography column was selected, based on the amount of available material. Extracts that were available at amounts of approximately 1g after solvent removal were fractionated on a 3x20cm
9
column. For extracts of 3-5g, a 5x35cm column was employed. The column was slurry packed with silica gel in hexane. Extracts which massed much less than 1g after solvent removal were not subjected to this procedure. In those cases, preparative thin layer chromatography was conducted.
Dissolving of the extract was attempted in several solvents, to find the best solubility. The extract was then dissolved and filtered if necessary to remove solids. A small aliquot was put aside, and the bulk of the solute was then absorbed into silica gel.
The resultant mixture of silica gel and solvent was then subjected to rotary evaporation to remove as much solvent as possible. Care was required for this procedure, to avoid bumping of the mixture and the resultant loss of material.
The resultant prepared mixture was added dry to the top of the column, and a
1cm layer of dry sand (Sigma-Aldrich, Catalog # 274739) was added to protect the column bed from disturbances from the addition of solvent.
The elution of this column was begun with pure hexane. Typically, the solvent reservoir was pressurized with nitrogen gas to facilitate elution. The technique is sometimes referred to as “flash chromatography.” The eluent fractions were collected, and tested by thin layer chromatography (TLC) as described below. The TLC results were used to determine when the elution of components was completed, and what stronger solvent system would be subsequently employed. Elution was conducted with hexane, mixtures of hexane and ethyl acetate, ethyl acetate, mixtures of ethyl acetate and methanol and finally methanol. Extracts that exhibited no spots by TLC, and which thus indicated no presence of eluted components, were discarded. Extracts indicating
10
the presence of single compounds by TLC were submitted for biological testing, while extracts indicating the presence of multiple components by TLC were subjected to preparative thin layer chromatography (preparative TLC.)
Thin Layer Chromatography. Thin layer chromatography (TLC) was conducted using silica on glass TLC plates (EM Science, Catalog # 5715-7.) These plates were infused by the manufacturer with chemicals that fluoresce when subjected to ultraviolet light at 254nm. Since aromatic compounds absorb light at 254nm, the presence of an aromatic compound on the plate would be detected as a blocking of the usual fluorescence. This phenomenon usually appears as a colored spot on the plate.
The plates were cut to approximately three centimeters by seven centimeters and spotted with the crude extract that had been saved from adsorption onto silica gel.
Several solvent systems, usually hexane, ethyl acetate, chloroform, methanol or mixtures of two or more of these solvents, are tried, to find the solvent system that gives the best separation of compounds in the crude extract.
Fresh plates were then spotted with the fractions from the chromatography column, in parallel with a sample from the original, crude extract. The plates were eluted with the solvent system selected during the original fractionation the crude extract.
Each plate was then viewed under ultraviolet light at 254nm, and the spots from aromatic compounds were circled. The TLC plates were then immersed in a solution of approximately one percent sulfuric acid in ethanol and baked with a heat gun to develop the spots from UV inactive compounds.18,19
11
Preparative Thin Layer Chromatgraphy. Preparative thin layer chromatography is conducted similarly to conventional TLC, except that the sample is spotted along a long line over the entire 20x20cm plate, or over several plates, depending upon the amount of material available. Also, the plates are not developed by immersion in ethanolic sulfuric acid, as this would destroy the components that are being isolated. This may lead to the loss of non-aromatic analytes, which would not absorb UV light at 254nm. However, most analytes will have some aromaticity, or unsaturations that will give detectable bands under UV light. The plates are eluted several times, using first pure hexane, and then increasing strengths of hexane: ethyl acetate to achieve a maximum separation of components.
When viewing under ultraviolet light indicates that the components are separated and/or approaching the top of the plate, the separate components are isolated as bands. The bands are harvested from the plate with a razor blade, and the mixture of silica gel and environmental component are loaded onto a 1x20cm chromatography column and eluted with dichloromethane. The resulting pure compound was concentrated by rotary evaporation and submitted for activity testing.
12
Chapter III
EXTRACTION PARAMETERS FOR SPECIFIC SAMPLES
Following is a detailed accounting of the extraction and fractionation of each
plant. These parameters may be followed to reproduce any of the results reported in this document.
Eriogonum brevicaule Nuttall. The samples of Eriogonum brevicaule Nuttall
(Family Polygonaceae) were submitted by Colorado State University. American Indians
used this plant, commonly known as buckwheat, as a lotion for skin cuts, in the
preparation of buffalo and deer hides, and as food.20 It was found to contain alkaloids,
making it a good species to investigate.21 The extraction was begun by placing 54.6g of
eriogonum leaves into a 4L Erlenmeyer flask with 1.0L hexane. The flask was swirled, and left standing for 72 hours. Then the solvent was isolated by filtration, yielding a
yellow extract, which was concentrated by rotary evaporation, to yield 0.63g dark
green/brown viscous liquid. This was called fraction AH1 for the purposes of activity
testing.
Into the flask with the remaining leaves was placed 1.0L ethyl acetate. This
extraction was allowed to proceed for 24 hours. The solvent was again isolated by
filtration and concentrated by rotary evaporation, yielding 0.30g dark green liquid.
(Fraction AH7.)
13
A third extraction was conducted on the remaining leaves, using 1.0.L ethanol.
Extraction proceeded for 48 hours, followed by filtration to isolate the extract. The filter cake was discarded. Rotary evaporation was difficult, as the concentrated extract became thick and bumping occurred. Therefore the extract was reduced to a volume of approximately 90mL, placed in a beaker, and allowed to air dry for 96 hours. This yielded 6.2g dark brown viscous liquid. (Fraction AH13.) At this point, the extract appeared dry and no solvent odor was detected.
A 0.34g aliquot of the hexane extract, fraction AH1, was suspended in acetone, and silica gel was added until a freely flowing olive green powder was obtained. A
350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 100mL ethyl acetate. The silica fraction was then poured on top of the rinsed silica and the cake was eluted with 4x50mL hexane. The final 50mL portion yielded an extract of little or no color, which was the criterion for completion of the fraction. All hexane elutions were combined and concentrated by rotary evaporation. This was called fraction
AH2 for the purposes of activity testing.
Similarly, the funnel was eluted with 6x50mL 1:1 hexane: ethyl acetate (Fraction
AH3), 4x50mL ethyl acetate, 4x50mL 1:1 ethyl acetate: methanol (Fraction AH5), and
4x50mL methanol (Fraction AH6.) Upon concentration by rotary evaporation, the ethyl acetate elution yielded too little material to be isolated. Therefore, there was no fraction
AH4.
A 0.09g aliquot of the ethyl acetate extract, fraction AH7, was suspended in acetone, and silica gel was added until a freely flowing olive green powder was obtained.
A 350mL glass fritted buchner funnel was loaded with approximately 100g silica and
14
rinsed with 100mL hexane. The silica fraction was then poured on top of the rinsed silica and the cake was eluted with 3x50mL hexane. No color was observed in any of these fractions. All hexane elutions were combined and concentrated by rotary evaporation, yielding no material whatsoever. Therefore, no fraction AH8 was isolated.
Similarly, the funnel was eluted with 6x50mL 1:1 hexane: ethyl acetate (Fraction
AH9), 4x50mL ethyl acetate (Fraction AH10), 4x50mL 1:1 ethyl acetate: methanol
(Fraction AH11), and 4x50mL methanol (Fraction AH12.)
A 3.03g aliquot of the ethanol extract, fraction AH13, was suspended in acetone, and silica gel was added until a freely flowing olive green powder was obtained. A
350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 100mL hexane. The silica fraction was then poured on top of the rinsed silica. Due to the large amount of extract, the silica fraction created a layer approximately 0.5cm thick. The cake was eluted with 5x50mL hexane. The final 50mL portion yielded an extract of little or no color, which was the criterion for completion of the fraction. All hexane elutions were combined and concentrated by rotary evaporation. This was called fraction AH14 for the purposes of activity testing.
Similarly, the funnel was eluted with 6x50mL 1:1 hexane: ethyl acetate (Fraction
AH15), 6x50mL ethyl acetate (Fraction AH16), 7x50mL 1:1 ethyl acetate: methanol
(Fraction AH17), and 6x50mL methanol (Fraction AH18.)
A second extraction of the same sample was begun by placing 127g of eriogonum leaves into a 4L Erlenmeyer flask with 1.0L hexane. The flask was swirled, and left standing for 72 hours. Then the solvent was isolated by filtration. This was concentrated by rotary evaporation, yielding 1.1g dark viscous liquid.
15
Into the flask with the remaining leaves was placed 1.0L ethyl acetate. This
extraction was allowed to proceed for 96 hours. The solvent was again isolated by
filtration and concentrated by rotary evaporation, yielding 0.92g.
The usual third extraction with methanol was not conducted, because none of the ethanol extracts from the original experiment demonstrated MDRP inhibition activity.
An aliquot of the hexane extract was suspended in acetone, and silica gel was
added until a freely flowing powder was obtained. A 350mL glass fritted buchner funnel
was loaded with approximately 100g silica and rinsed with 100mL hexane. The silica
fraction was then poured on top of the rinsed silica and the cake was eluted with 5x50mL
hexane. All hexane elutions were combined and concentrated by rotary evaporation.
This was called fraction AH19 for the purposes of activity testing.
Similarly, the funnel was eluted with 5x50mL each of 1:1 hexane: ethyl acetate,
ethyl acetate, 1:1 ethyl acetate: methanol, and methanol. None of these extracts were submitted for testing, because they demonstrated no activity in the original testing.
An aliquot of the ethyl acetate extract was suspended in acetone, and silica gel
was added until a freely flowing powder was obtained. A 350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 100mL hexane. The
silica fraction was then poured on top of the rinsed silica and the cake was eluted with
5x50mL hexane. No color was observed in any of these fractions.
Similarly, the funnel was eluted with 5x50mL each of 1:1 hexane: ethyl acetate, ethyl acetate, 1:1 ethyl acetate: methanol, and methanol (Fraction AH21.) Only the methanol extract was submitted for testing, because this was the only fraction that demonstrated activity in the original testing.
16
Additional Eriogonum Samples. Dr. Frank Stermitz of Colorado State
University provided five additional eriogonum samples. These samples were labeled
“Leaves”, “Roots”, “Hillside”, “Field by Cement Plant” and “Eriogonum.” The last two samples appeared to be identical to the originally provided sample, except that they were collected at different times and different locations. These samples were extracted similarly to the original eriogonum extract, as outlined in Table 1.
With this extraction and the following, a new procedure was used for the final concentration of many of the samples. Completing the entire concentration by rotary evaporation was difficult, because bumping often occurred, which lead to loss of material and the risk of cross contamination. Therefore, the fractions were concentrated to convenient volumes of 20mL or less, and transferred to 20mL scintillation vials. The vials were then placed in a laboratory hood overnight, to allow airflow to evaporate the solvent slowly and gently. This yielded good concentrated fractions with no observable solvent remaining.
17
Table 1. Eriogonum Hillside Extraction and Fractionation Parameters Hillside Sample Extraction Parameters Original mass 112.7g Yields/ ID’s Hexane extraction time 48 hr 0.94g/ AH71 Ethyl acetate extraction time 24 hr 1.38g/ AH77 Methanol extraction time 24 hr 15.6g/ AH83
Hillside Sample Fractionation Parameters/ ID’s Elution Hexane 50:50 hexane: ethyl acetate 50:50 ethyl methanol ethyl acetate acetate:methanol volumes Hexane 4x50mL/AH72 4x50mL/AH73 4x50mL/AH74 4x50mL/AH75 4x50mL/AH76 Ethyl acetate 4x50mL/none 5x50mL/AH79 5x50mL/AH80 5x50mL/AH81 5x50mL/AH82 Methanol 6x50mL/none 6x50mL/AH85 6x50mL/AH86 6x50mL/AH87 6x50mL/AH88
Hillside Sample Fraction Appearance Yields Hexane 50:50 hexane: Ethyl acetate 50:50 ethyl methanol ethyl acetate acetate:methanol Hexane no color dark green Gold yellow light yellow Ethyl acetate no color dark green Gold gold yellow Methanol no color green/brown Yellow brown/turbid brown
The “Field by Cement Plant” (FxCP) sample was extracted and fractionated with the assistance of Mark Nikbakht, an undergraduate student at Southern Methodist
University. This student has since graduated, and his research notebooks could not be located. Therefore, some data points were lost. The available data are outlined in
Table 2.
18
Table 2. Eriogonum Field by Cement Plant Extraction and Fractionation Parameters Field by Cement Plant Sample Extraction ID’s Hexane extraction AH125 Ethyl acetate extraction AH131 Methanol extraction AH137
Field by Cement Plant Sample Fraction ID’s Elution Hexane 50:50 hexane: ethyl acetate 50:50 ethyl methanol ethyl acetate acetate:methanol volumes Hexane AH126 AH127 AH128 AH129 AH130 Ethyl acetate None AH133 AH134 AH135 AH136 Methanol None none None AH141 AH142
Field by Cement Plant Sample Fraction Appearance Yields Hexane 50:50 hexane: Ethyl acetate 50:50 ethyl methanol ethyl acetate acetate:methanol Methanol no color no color no color dark brown dark brown
Table 3. Eriogonum Leaves Extraction and Fractionation Parameters Leaves Sample Extraction Parameters Original mass 74.2g Yields/ ID’s Hexane extraction time 24 hr 0.94g/ AH89 Ethyl acetate extraction time 48 hr 2.00g/ AH95 Methanol extraction time 48 hr 7.55g/ AH101
Leaves Sample Fractionation Parameters/ ID’s Elution Hexane 50:50 hexane: ethyl ethyl acetate 50:50 ethyl methanol acetate acetate:methanol volumes Hexane 4x50mL/AH90 6x50mL/AH91 5x50mL/AH92 4x50mL/AH93 4x50mL/AH94 Ethyl acetate 5x50mL/none 6x50mL/AH97 5x50mL/AH98 4x50mL/AH99 5x50mL/AH100 Methanol 4x50mL/none 6x50mL/AH103 5x50mL/AH104 6x50mL/AH105 6x50mL/AH106
Leaves Sample Fraction Appearance Yields Hexane 50:50 hexane: Ethyl acetate 50:50 ethyl methanol ethyl acetate acetate:methanol Hexane no color yellow/green Yellow light yellow light yellow Ethyl acetate no color yellow/green Yellow light yellow light yellow Methanol no color yellow/green Yellow/green yellow/turbid yellow/brown 19
Table 4. Eriogonum Roots Extraction and Fractionation Parameters Roots Sample Extraction Parameters Original mass 21.7g Yields/ ID’s Hexane extraction time 48 hr 0.18g/ AH107 Ethyl acetate extraction time 48 hr 0.29g/ AH113 Methanol extraction time 24 hr 1.14g/ AH119
Roots Sample Fractionation Parameters/ ID’s Elution Hexane 50:50 hexane: ethyl ethyl acetate 50:50 ethyl Methanol acetate acetate:methanol volumes Hexane 3x50mL/none 4x50mL/AH109 6x50mL/AH110 4x50mL/AH111 4x50mL/AH112 Ethyl acetate 4x50mL/none 4x50mL/AH115 6x50mL/AH116 4x50mL/AH117 4x50mL/AH118 Methanol 4x50mL/none 6x50mL/none 6x50mL/none 6x50ml/AH123 8x50mL/AH124
Roots Sample Fraction Appearance Yields Hexane 50:50 hexane: Ethyl acetate 50:50 ethyl Methanol ethyl acetate acetate:methanol Hexane light yellow yellow/green light brown no color no color Ethyl acetate no color light yellow Yellow/brown light brown light brown Methanol no color light brown light brown light brown light brown
20
Table 5. Eriogonum Re-extraction and Fractionation Parameters Eriogonum Sample Extraction Parameters Original mass 220.8g Yields/ ID’s Hexane extraction time 24 hr 2g/ AH53 Ethyl acetate extraction time 96 hr 2.89g/ AH59 Methanol extraction time 24 hr 10.4g/ AH65
Eriogonum Sample Fractionation Parameters/ ID’s Elution Hexane 50:50 hexane: ethyl acetate 50:50 ethyl Methanol ethyl acetate acetate:methanol volumes Hexane 8x50mL/AH54 6x50mL/AH55 6x50mL/AH56 6x50mL/AH57 5x50mL/AH58 Ethyl acetate 4x50mL/AH60 6x50mL/AH61 6x50mL/AH62 6x50mL/AH63 5x50mL/AH64 Methanol 4x50mL/none 6x50mL/AH67 6x50mL/AH68 6x50mL/AH69 5x50mL/AH70
Eriogonum Sample Fraction Appearance Yields Hexane 50:50 hexane: Ethyl acetate 50:50 ethyl Methanol ethyl acetate acetate:methanol Hexane no color yellow/green dark green yellow/green light yellow Ethyl acetate no color yellow/green dark green dark green yellow/green Methanol no color yellow/green dark green green/brown yellow/brown
Silica Clean-up of Active Fractions. Additional silica clean up was conducted for fractions AH19 and AH21. In each case, approximately 30g silica was slurry packed into a 20x500mm liquid chromatography column. The sample extracts were separately dissolved in 2mL hexane and charged onto the silica column. The column was eluted with 80:20 hexane: ethyl acetate, and 10mL aliquots were collected. Each fraction was analyzed by thin layer chromatography (TLC), eluting with 80:20 hexane: ethyl acetate and developing with ethanolic sulfuric acid.
Based on the TLC results for the clean up of fraction AH19, silica fractions 4-13 were combined, solvent removed by rotary evaporation, and the resultant material was subjected to MDRP inhibitor testing (Fraction AH20.) For AH21, silica fractions 1-20
21
were combined, solvent removed by rotary evaporation, and the resultant material was subjected to MDRP inhibitor testing (Fraction AH22.)
Silica Fractionation of Active Fractions. Fractions AH103 and AH127 demonstrated MDRP inhibition activity, and fraction AH73 demonstrated less desirable direct activity.
All three extracts were dissolved in methanol and analyzed by TLC, eluting with
80:20 hexane: ethyl acetate. The resultant plates displayed no compounds by UV fluorescence. A developing solution was created by adding 2 drops of concentrated sulfuric acid to 500mL 95% ethanol. The plates were then developed by emersion in the dilute ethanolic sulfuric acid, and heating with a heat gun.
The three extracts exhibited similar TLC results, and their combined mass totaled less than 1.5g material. Therefore, the three fractions were combined for silica liquid chromatography fractionation.
The column was prepared by slurry packing a 3x200cm liquid chromatography column to a height of 170cm. Excess solvent was allowed to drain. The sample was prepared by combining the three extracts, AH73, AH103 and AH127, removing excess methanol by rotary evaporation, adding silica to create a free-flowing powder, and rotary evaporating again. This extract was charged onto the top of the packing, and a small amount of clean sand was added to protect the packing bed from disturbance as solvents were added. The column was then eluted as outlined in Table 6.
Preparative Thin Layer Chromatography of Active Fractions. Fraction AH64 was reported to exhibit remarkable activity. Therefore, the sample was further fractionated by preparative thin layer chromatography. The extract (25mg) was dissolved
22
in 1mL of methanol and spotted across two glass TLC plates. The plates were eluted with
50:50 hexane: ethyl acetate, 98:2 chloroform: methanol, and twice with 95:5
Table 6. Flash Chromatography Parameters for AH73/AH103/AH127 Fractions Solvent Volume/Fraction Fraction Rinses hexane 200 mL discard 1-9 125 mL 10-35 50 mL 36-49 20 mL 50-56 50 mL 57-110 99:1 hexane: 50 mL ethyl acetate 111-115 50 mL AH211 116-120 50 mL 121-125 50 mL AH212 126-128 50 mL 129-148 95:5 hexane: 50 mL ethyl acetate 149-157 50 mL AH213 158-165 50 mL AH214 166-176 50 mL 177-187 50 mL 188-195 90:10 hexane: 50 mL AH215 ethyl acetate 196-217 50 mL AH216 218-233 50 mL 234-245 50 mL AH217 246-273 50 mL AH218 274-282 50 mL 283-298 80:20 hexane: 50 mL ethyl acetate 299-308 50 mL AH219 326-340 50:50 hexane: 100 mL AH220 ethyl acetate 341-360 100 mL
chloroform: methanol. This yielded, top to bottom, a pink band (AH270), a dark brown band (AH271), a second brown band (AH272), and a band of stationary material. The three bands were harvested and isolated for MDRP inhibition testing.
23
The band of stationary material was isolated, re-dissolved, and re-applied to two
TLC plates. This material was eluted with 80:20 chloroform: methanol. The yielded, top
to bottom, a brown band (AH273) and a dark brown band (AH274.) These two bands
were harvested and isolated for testing.
Hydrastis canadensis. The common name for Hydrastis canadensis is goldenseal, and it is available in most drug stores. Native Americans used this plant to treat indigestion, diarrhea, fever, wounds and local inflammation. Some U.S. physicians use plant as an antiseptic, astringent, and an anti-inflammatory.22 It has been found to
naturally contain the antibiotic berberine.23
The extraction of this plant was begun by placing 113.7g of goldenseal leaves into
a 4L Erlenmeyer flask with 1.0L hexane. The flask was swirled, and left standing for 48
hours. Then the solvent was isolated by filtration and concentrated by rotary evaporation, yielding 1.34g dark green liquid. This was called fraction AH23 for the purposes of activity testing.
Into the flask with the remaining leaves was placed 1.0L ethyl acetate. This extraction was allowed to proceed for 24 hours. The solvent was again isolated be filtration and concentrated by rotary evaporation, yielding 3.21g bright green liquid.
(Fraction AH29.)
A third extraction was conducted on the remaining leaves, using 1.0L methanol.
Extraction proceeded for 24 hours, followed by filtration to isolate the extract. The filter cake was discarded. This filtrate was concentrated, yielding 14.8g dark brown viscous liquid. (Fraction AH35.)
24
A 0.42g aliquot of the hexane extract, fraction AH23, was fractionated by vacuum
liquid chromatography as previously described. The cake was eluted with 4x50mL
hexane yielding extracts of little or no color. A yellow-orange band traveled slowly
down the column, but did not elute. All hexane elutions were combined and concentrated, yielding no observable material. Therefore, there was no fraction AH24.
Similarly, the funnel was eluted with 5x50mL 1:1 hexane:ethyl acetate (Fraction
AH25), yielding the yellow-orange band and dark green material. The funnel was eluted
with 4x50mL ethyl acetate(Fraction AH26), 4x50mL 1:1 ethyl acetate: methanol
(Fraction AH27), and 5x50mL methanol. Upon concentration by rotary evaporation,
AH26 and AH27 yielded yellow-orange and yellow extracts, respectively. The methanol elution yielded too little material to be isolated. Hence, there was no fraction AH28.
A 2.0g aliquot of the ethyl acetate extract, fraction AH29, was also analyzed by
VLC. The silica column was eluted with 6x50mL hexane yielding extracts of little or no color. A yellow band traveled slowly down the column, but did not elute. All hexane elutions were combined and concentrated, yielding no observable material. Therefore, there was no fraction AH30.
Similarly, the funnel was eluted with 8x50mL 1:1 hexane:ethyl acetate (Fraction
AH31), 6x50mL ethyl acetate (Fraction AH32), 6x50mL 1:1 ethyl acetate: methanol
(Fraction AH33), and 6x50mL methanol (Fraction AH34.)
A 7.4g aliquot of the methanol extract, fraction AH35, was analyzed by VLC.
The first elution was 8x50mL hexane, which eluted and isolated the yellow band observed in the other two extracts (Fraction AH36.) The final extract was yellow in color.
25
Similarly, the funnel was eluted with 8x50mL 1:1 hexane: ethyl acetate (Fraction
AH37), 6x50mL ethyl acetate (Fraction AH38), 6x50mL 1:1 ethyl acetate: methanol
(Fraction AH39), and 6x50mL methanol (Fraction AH40.) Fraction AH37 yielded a dark
green extract upon rotary evaporation, and the others yielded green-brown extracts.
Biehl #45. Dr. Edward Biehl completed the initial solvent extraction of this
Hydrastis canadensis stems and branches sample. A 0.51g aliquot of methanol extract was presented for fractionation and analysis.
The aliquot, fraction AH41, was suspended in acetone, and silica gel was added until a freely flowing powder was obtained. A 350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 100mL hexane. The silica fraction was then poured on top of the rinsed silica. The cake was eluted with 5x50mL hexane, yielding extracts of no color. All hexane elutions were combined and concentrated by rotary evaporation, yielding no observable material. Therefore, there was no fraction
AH42.
Similarly, the funnel was eluted with 6x50mL 1:1 hexane: ethyl acetate. This was called fraction AH43 for the purposes of activity testing. Elutions continued with
6x50mL ethyl acetate (Fraction AH44), 7x50mL 1:1 ethyl acetate: methanol (Fraction
AH45), and 6x50mL methanol (Fraction AH46.) These fractions were dark green, yellow-green, green and yellow-green, respectively.
Biehl #246. Dr. Edward Biehl completed the initial solvent extraction of this
Hydrastis canadensis sample. A 0.39g aliquot of extract was presented for fractionation and analysis.
26
The aliquot, fraction AH47, was suspended in acetone, and silica gel was added
until a freely flowing powder was obtained. A 350mL glass fritted buchner funnel was
loaded with approximately 100g silica and rinsed with 100mL hexane. The silica fraction
was then poured on top of the rinsed silica. The cake was eluted with 6x50mL hexane,
yielding extracts of no color. All hexane elutions were combined and concentrated by
rotary evaporation. This was called fraction AH48 for the purposes of activity testing.
Similarly, the funnel was eluted with 8x50mL 1:1 hexane: ethyl acetate (Fraction
AH49), 6x50mL ethyl acetate (Fraction AH50), 6x50mL 1:1 ethyl acetate: methanol
(Fraction AH51), and 6x50mL methanol (Fraction AH52.) These fractions were black,
bright green, bright green and yellow, respectively.
Pouteria pallida - N014795. This sample of Pouteria pallida was delivered as an
extract from Dr. Kim Lewis at Northeastern University. Therefore, extraction and
vacuum liquid chromatography was not conducted at Southern Methodist University.
Pouteria pallida, known as balata, produces fruit which is edible, although not
popular.24,25 Silica fractionation was conducted to isolate compounds for multi-drug
resistance pump inhibition activity.
Silica Fractionation. The extract was dissolved in a one to one mixture ethyl
acetate and methanol and analyzed by TLC, eluting with 50:50 hexane: ethyl acetate.
The resultant plates displayed five compounds by UV fluorescence. The plates were then
developed using dilute ethanolic sulfuric acid, as previously described, yielding two
additional spots.
A flash chromatography column was prepared by slurry packing a 5x35 cm liquid chromatography column to a height of 20cm. Excess solvent was allowed to drain. The
27
sample was prepared by dissolving in a 1:1 mixture of ethyl acetate: methanol. Acetone
was added to dissolve insoluble residues, and the final solution was filtered to isolate
traces of insoluble material. The resultant filtrate was concentrated by rotary evaporation
to approximately 20 milliliters. Approximately 10 milliliters of silica was added to create
a free-flowing powder, and the remaining solvent was removed by rotary evaporation.
This extract was charged onto the top of the packing, and a small amount of clean sand
was added to protect the packing bed from disturbance as solvents were added. The
column was then eluted as outlined in Table 7.
Table 7. Flash Chromatography Parameters for N014795 Pouteria pallida Fractions Solvent Volume/Fraction Fraction Rinses hexane 1500 mL Discard 1-28 95:5 hexane: 125 mL ethyl acetate 29-36 125 mL “A”, also AH228 37-40 125 mL Discard 41-47 125 mL AH202 48-64 125 mL “C” 65-80 90:10 hexane: 125 mL Discard ethyl acetate 81-100 83:17 hexane: 125 mL Discard ethyl acetate 101-104 80:20 hexane: 125 mL Discard ethyl acetate 105-126 125 mL AH206 127-135 125 mL AH207 136-150 50:50 hexane: 125 mL AH208 ethyl acetate 151-163 125 mL AH209 164-174 ethyl acetate 125 mL AH210 175-204 125 mL AH229 205 80:20 ethyl 125 mL AH230 acetate:methanol 206-208 125 mL AH231 209-212 125 mL AH232 213-216 methanol 125 mL AH233
28
Preparatory Thin Layer Chromatography. The fractions designated “A” and
“C” exhibited multiple peaks by thin layer chromatography, and therefore was further
separated by preparatory thin layer chromatography (prep TLC.)
Fraction “A” was dissolved in ethyl acetate and spotted across two 20x20cm silica
TLC plates, and eluted nine times with 95:5 hexane: ethyl acetate. The mobile phase was allowed to elute to the top of the plate each time. This yielded four colored elution bands and a band of material that did not travel. The four bands were removed with a razor blade, and the isolated silica was placed in a 1x15cm chromatography column and eluted with ethyl acetate. One of these bands yielded a purified, white solid, which was submitted for activity testing (AH201.)
Fraction “C” was dissolved in dichloromethane and spotted across five 20x20cm silica TLC plates, and eluted multiple times with 90:10 hexane: ethyl acetate. The mobile phase was allowed to elute to the top of the plate each time. This yielded five colored elution bands and a band of material that did not travel. The five bands were removed with a razor blade, and the isolated silica was placed in a 1x15cm chromatography column and eluted with dichloromethane. Two of these bands yielded a yellow, oily material, and a third yielded a purified, white solid. These three extracts were submitted for activity testing (AH203, AH204 and AH205.)
The fraction AH207 demonstrated biological activity, and was further fractionated by preparatory TLC. This fractionation was conducted by Dr. Ramadas Sathunuru, and yielded fractions AH221 through AH 227, which were submitted for activity testing.
Thuja occidentalis - N102279. This sample of Thuja occidentalis was delivered as an extract from Dr. Kim Lewis at Northeastern University. Therefore, extraction and
29
vacuum liquid chromatography was not conducted at Southern Methodist University.
The common name of this plant is white cedar, and it is endemic to eastern North
America.26 Extracts of this plant have been shown to have antiviral activity.27 Silica
fractionation was conducted to isolate compounds for multi-drug resistance pump
inhibition activity.
Silica Fractionation. Approximately 20mg of the extract was dissolved in a one
to one mixture ethyl acetate and methanol and analyzed by TLC, eluting with chloroform,
7:2 hexane: ethyl acetate and finally 1:1 hexane: ethyl acetate, as the separation
progressed. The extract contained some polar compounds that required the stronger
solvents for elution to occur. The resultant plates displayed six compounds by UV
fluorescence. The plates were then developed using dilute ethanolic sulfuric acid, as
previously described, yielding one additional spot.
The column was prepared by slurry packing a 5x35 cm liquid chromatography
column to a height of 20cm. Excess solvent was allowed to drain. The sample was
prepared by dissolving in a 1:1 mixture of ethyl acetate: methanol. Approximately 60
milliliters of silica was added, and the remaining solvent was removed by rotary
evaporation, to create a free flowing powder. This extract was charged onto the top of
the packing, and a small amount of clean sand was added to protect the packing bed from
disturbance as solvents were added. The column was then eluted as specified in Table 8.
Preparatory Thin Layer Chromatography. The combined fractions 10 through
12 exhibited two spots by thin layer chromatography, and therefore were further
separated by preparatory thin layer chromatography (TLC.)
30
This fraction was dissolved in ethyl acetate and spotted across six 20x20cm silica
TLC plates, and eluted four times with hexane, once with 99:1 hexane: ethyl acetate, and
five times with 95:5 hexane: ethyl acetate. The mobile phase was allowed to elute to the
top of the plate each time. This yielded two colored elution bands. The two bands were
removed with a razor blade, and the isolated silica was placed in a 1x15cm
chromatography column and eluted with ethyl acetate. The two bands yielded white oils,
which were submitted for activity testing (AH234, AH235.)
Table 8. Flash Chromatography Parameters for N102279 Thuja occidentalis Fractions Solvent Volume/Fraction Fraction Rinses hexane 2000 mL discard 1-3 600 mL discard 4-9 90:10 hexane: 125 mL discard ethyl acetate 10-12 125 mL fractionate 13-14 125 mL AH236 15-19 50 mL AH236 20-24 125 mL AH236 25-29 70:30 hexane: 125 mL AH236 ethyl acetate 30-32 125 mL AH237 33-36 125 mL AH238 37-38 125 mL AH239 39-40 50:50 hexane: 125 mL AH239 ethyl acetate 41-42 125 mL discard 43-44 125 mL AH240 45-63 125 mL AH241 64-76 ethyl acetate 125 mL AH242
Thuja occidentalis - N102249. This sample of Thuja occidentalis was delivered
as an extract from Dr. Kim Lewis at Northeastern University. This fraction was extracted
31
by vacuum liquid chromatography to isolate compounds for multi-drug resistance pump
inhibition activity. This was at variance to the procedure conducted on Thuja
occidentalis sample N102279, which was fractionated by flash liquid chromatography.
Vacuum Liquid Chromatography. A 3.70g aliquot of the extract was suspended in 1:1 ethyl acetate: methanol, and silica gel was added until a freely flowing rosy pink powder was obtained. A 350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 200mL hexane. The silica fraction was then poured on top of the rinsed silica and the cake was eluted with 3x100mL hexane. The final 100mL portion yielded a colorless extract, which was the criterion for completion of the fraction. All hexane elutions were combined and concentrated by rotary evaporation.
This yielded too little material to be isolated. Therefore, there was no fraction AH243 for the purposes of activity testing.
Similarly, the funnel was eluted with 5x100mL 1:1 hexane: ethyl acetate (Fraction
AH244), 6x100mL ethyl acetate (Fraction AH245), 4x100mL 1:1 ethyl acetate: methanol
(Fraction AH246), and 4x100mL methanol (Fraction AH247.) Each of these extracts was submitted for activity testing.
Rhus trilobata - N102791. This sample of Rhus trilobata was delivered as an extract from Dr. Kim Lewis at Northeastern University. The common name for this plant is skunk bush or squaw bush.28 It has been considered, as an ozone-sensitive species, for
ozone monitoring.29 This fraction was extracted by vacuum liquid chromatography to isolate compounds for multi-drug resistance pump inhibition activity.
Vacuum Liquid Chromatography. A 4.16g aliquot of the extract was suspended in 1:1 ethyl acetate: methanol, and silica gel was added until a freely flowing
32
rosy pink powder was obtained. A 350mL glass fritted buchner funnel was loaded with
approximately 100g silica and rinsed with 200mL hexane. The silica fraction was then
poured on top of the rinsed silica and the cake was eluted with 3x100mL hexane. The final 100mL portion yielded a colorless extract, which was the criterion for completion of the fraction. All hexane elutions were combined and concentrated by rotary evaporation.
This yielded too little material to be isolated. Therefore, there was no fraction AH248 for the purposes of activity testing.
Similarly, the funnel was eluted with 100mL 9:1 hexane: ethyl acetate and
2x100mL 8:2 hexane:ethyl acetate (combined to create Fraction AH249), 5x50mL 8:2 ethyl acetate: methanol (Fraction AH250) 3x100mL ethyl acetate (Fraction AH251),
4x100mL 1:1 ethyl acetate:methanol (Fraction AH252), and 4x100mL methanol
(Fraction AH253.) Each of these extracts was submitted for activity testing.
Sample AH 250 demonstrated positive MDRP inhibition activity. Therefore, it was further fractionated by liquid chromatography, and the sub-fractions were submitted for further testing.
Silica Fractionation. Approximately 20mg of the extract was dissolved in ethyl
acetate and analyzed by TLC, eluting with 80:20 hexane: ethyl acetate. The resultant
plates displayed three compounds by UV fluorescence. The plates were then developed
using dilute ethanolic sulfuric acid, as previously described, yielding two additional
spots.
A flash chromatography column was prepared by dry packing a 2.5x50 cm liquid
chromatography column to a height of 20cm. The column was charged with hexane, and
excess solvent was allowed to drain. The 200mg sample was prepared by dissolving in
33
approximately 5mL ethyl acetate. Three hundred ninety milligrams of silica was added,
and the solvent was removed by rotary evaporation, to create a free flowing powder.
Table 9. Flash Chromatography Parameters for Rhus trilobata AH250
Fractions Solvent Volume/Fraction Fraction Rinses hexane 200 mL discard 1-14 99:1 hexane: 50 mL discard ethyl acetate 15-20 98:2 hexane: 50 mL discard ethyl acetate 21-52 95:5 hexane: 50 mL AH261 ethyl acetate 53-68 50 mL AH262 69-72 50 mL discard 73-96 50 mL AH263 97-101 90:10 hexane: 50 mL discard ethyl acetate 102-110 50 mL AH264 111-115 50 mL discard 116-121 50 mL AH265 122-123 50 mL discard 124-137 50 mL AH266 138-151 80:20 hexane: 50 mL AH267 ethyl acetate 152-155 50:50 hexane: 50 mL AH268 ethyl acetate 156-162 50 mL discard 163-168 ethyl acetate 50 mL AH269
This extract was charged onto the top of the packing, and a small amount of clean
sand was added to protect the packing bed from disturbance as solvents are added. The
column was then eluted as specified in Table 9. The resultant extracts were submitted for
MDRP inhibition testing.
34
Gunnera macrophylla - N014045. This sample of Gunnera macrophylla was
delivered as an extract from Dr. Kim Lewis at Northeastern University. This plant is
described as a “sort of large-leaved Forget-me-not”30, and is most noted in botanical
literature for its symbiotic relationship with the nitrogen-fixing blue-green alga Nostoc
muscorum.31,32 This fraction was extracted and vacuum liquid chromatography to isolate
compounds for multi-drug resistance pump inhibition activity.
Vacuum Liquid Chromatography. A 4.78g aliquot of the extract was
suspended in approximately 100mL 1:1 ethyl acetate: methanol. Gentle warming for one
half hour completed final dissolution. Silica gel was added, and the solvent was removed
by rotary evaporation, resulting in a freely flowing olive green powder. A 350mL glass fritted buchner funnel was loaded with approximately 100g silica and rinsed with 200mL
hexane. The silica fraction was then poured on top of the rinsed silica and the cake was
eluted with 100mL hexane. However, the solvent was found to be contaminated with a
ketone, probably acetone, and much of the organics were eluted. Therefore, this first
fraction was concentrated, and set aside for fractionation with good solvent.
The funnel was then eluted with 4x100mL ethyl acetate (Fraction AH257),
4x100mL 1:1 ethyl acetate: methanol (Fraction AH259), and 3x100mL methanol
(Fraction AH260.) Each of these extracts was submitted for activity testing.
The original extract eluted with contaminated hexane was concentrated, dissolved
in approximately 15mL ethyl acetate, and re-adsorbed onto silica gel as before. The
sample was then charged onto the 350mL fritted buchner funnel, and eluted with
4x100mL hexane. Final concentration of this extract yielded too little material to be
isolated. Therefore, there was no fraction AH254 for the purposes of activity testing.
35
Similarly, the funnel was eluted with 3x100mL 9:1 hexane: ethyl acetate (Fraction
AH255), 4x100mL 1:1 hexane:ethyl acetate (Fraction AH256), and 3x100mL ethyl acetate (Fraction AH258.) Each of these extracts was submitted for activity testing.
36
Chapter IV
MDRP ACTIVITY TESTING RESULTS
All of the fractions that were generated were submitted for multi-drug resistance pump inhibition activity testing. The fractions were extracted from plants that have clinically, or in traditional medicine, demonstrated useful resistance to bacteria, indicating the possible presence of multi-drug resistance pump inhibitors. Since the extractions were taken from the entire plant, it is reasonable that most of the resultant fractions will demonstrate no activity. The results obtained from all of the submitted samples are presented in Appendix B. The possible results from the testing include the following.
No Activity. This indicates that this extract had no positive or negative effect on the encroachment of the bacteria Staphylococcus aureus. There is no further need to investigate extracts that exhibit this result.
Direct Activity. This indicates that the extract inhibits the growth of the bacteria in the presence or absence of the antibiotic. This is not a useful result, because this indicates that the extract is directly poisonous to the bacteria. This may mean that the extract would also be poisonous to the host organism. In any case, MDRP inhibition activity is not demonstrated. It must be observed that many of these extracts contain multiple chemical species, and the presence of a MDRP inhibitor may be
37
masked by the presence of a direct poison. These extracts may be further fractionated in search of the useful inhibitory molecule. However, at the time of this publication, these extracts are being set aside for potential future study.
Slight Activity. Some extracts exhibit some reduction in bacteria growth at higher concentrations, but no complete inhibition at the tested concentrations. These extracts are considered on a case-by-case basis.
Positive Activity. These extracts show strong MDRP-inhibition activity at high and low concentrations. These are the most promising extracts, and are selected for further fractionation and isolation of individual compounds. In most cases, multiple fractions are generated which are submitted for activity testing in turn.
In this section, a detailed description of the fates of all extracts demonstrating positive or slight activity is provided. Data for the pertinent fractions are inserted.
Complete data for all fractions are included in Appendix C.
AH2 – Eriogonum brevicaule AH2 AH2 S. aureus 0.17 0.141 extract Nuttall /Hexane extract/100% Hexane 0.167 0.171 berberine 0.041 0.035 500 ug/mL ext + ber VLC fraction. This fraction (40mg) was 0.085 0.072 250 ug/mL 0.123 0.103 125 ug/mL consumed in activity testing, and 0.136 0.111 62.5 ug/mL 0.21 0.195 cells additional Eriogonum plants were 0.001 0.000 MHB extracted, yielding samples AH19 and AH54. Results for these fractions are pending.
38
AH6 – Eriogonum brevicaule AH 6 AH 6 S. aureus 0.192 0.193 extract Nuttall /Hexane extract/100% Methanol 0.206 0.203 berberine 0.055 0.057 500 ug/mL ext + ber VLC fraction. This fraction (7mg) was 0.066 0.111 250 ug/mL 0.102 0.167 125 ug/mL consumed in activity testing, and 0.139 0.214 62.5 ug/mL 0.258 0.326 cells additional Eriogonum plants were 0.000 0.001 MHB extracted, yielding sample AH58. Results for this fraction are pending.
AH12 – Eriogonum brevicaule AH 12 AH 12 S. aureus 0.225 0.181 extract Nuttall /Ethyl acetate extract/100% 0.19 0.215 berberine 0.091 0.071 500 ug/mL ext + ber Methanol VLC fraction. This fraction 0.101 0.087 250 ug/mL 0.09 0.136 125 ug/mL (3mg) was consumed in activity testing, 0.14 0.24 62.5 ug/mL 0.202 0.226 cells and additional Eriogonum plants were 0.001 0.000 MHB extracted, yielding samples AH21 and AH64. Results for these fractions are pending.
AH64 – Eriogonum brevicaule Nuttall /Ethyl acetate extract/100% Methanol
VLC fraction. This fraction (100mg) was reported to have remarkable activity.
Numerical results are pending. AH64 was further fractionated by preparative thin layer chromatography, yielding samples AH270 through AH274. Test results for this fraction are pending.
AH71 – Eriogonum brevicaule Nuttall AH71 S. aureus 0.1235 Extract /Hexane extract. This extract (940mg) exhibited 0.21595 Berberine 0.0089 500ug/ml ext + ber slight activity. The extract was fractionated by 0.0096 250ug/ml 0.1104 125ug/ml vacuum liquid chromatography to yield fractions 0.1403 62.5ug/ml 0.2462 Cells AH72 through AH76. These fractions exhibited no -0.00065 MHB
39
useful activity. Fraction AH 73 was fractionated further as noted below, with fraction
AH103.
AH81 – Eriogonum brevicaule Nuttall AH81 S. aureus 0.118 Extract (Hillside)/Ethyl acetate extract/50% Ethyl acetate: 0.2196 Berberine 0.0697 500ug/ml ext + ber Methanol VLC fraction. This fraction (450mg) 0.1009 250ug/ml 0.1698 125ug/ml exhibited very slight activity, and possible direct 0.2085 62.5ug/ml 0.22105 Cells activity. As the results showed very little activity, and -0.00035 MHB the fraction was tarry and complex, this fraction has not been pursued.
AH103 – Eriogonum brevicaule Nuttall AH103 S. aureus 0.11145 Extract (Leaves)/Methanol extract/50% Hexane:ethyl 0.2095 Berberine 0.088 50ug/ml ext + ber acetate VLC Fraction. This fraction (81mg), which 0.1959 25ug/ml 0.23325 12.5ug/ml exhibited slight activity, was compared by thin layer 0.2246 6.25ug/ml 0.22105 Cells chromatography to fractions AH73 and AH127, which -0.00085 MHB were also 50% Hexane: ethyl acetate VLC fractions of the Eriogonum plant. As these extracts were comparable by thin layer chromatography, they were combined to generate approximately 1.4g of material for silica liquid chromatography fractionation.
This fractionation yielded samples AH211 through AH220. Of these fractions, AH 218 exhibited slight activity. As the activity was slight, and the amount of remaining material was small, this extract was not pursued further.
40
AH107 – Eriogonum brevicaule Nuttall AH107 S. aureus 0.1621 Extract (Roots)/Hexane extract. This extract (180mg), which 0.14275 Berberine 0.0942 50ug/ml ext + ber exhibited slight activity, was fractionated by vacuum 0.03305 25ug/ml 0.16225 12.5ug/ml liquid chromatography to yield fractions AH109 0.2283 6.25ug/ml 0.2103 Cells through AH112. Results are pending for these -0.0051 MHB fractions.
AH127 – Eriogonum brevicaule Nuttall (Field AH127 S. aureus 0.16255 Extract by Cement Plant)/Hexane extract/50% Hexane:ethyl 0.2986 Berberine -0.0141 50ug/ml ext + ber acetate VLC Fraction. This fraction (800mg), which -0.0082 25ug/ml 0.17985 12.5ug/ml exhibited good MDRP-inhibitory activity, was 0.2512 6.25ug/ml 0.2675 Cells compared by thin layer chromatography to fractions -1.00E-04 MHB
AH73 and AH103, which were also 50% Hexane: ethyl acetate VLC fractions of the
Eriogonum plant. As these extracts were comparable by thin layer chromatography, they were combined to generate approximately 1.4g of material for silica liquid chromatography fractionation. This fractionation yielded samples AH211 through
AH220. Of these fractions, AH 218 exhibited slight activity. As the activity was slight, and the amount of remaining material was small, this extract was not pursued further.
AH133 – Eriogonum brevicaule Nuttall (Field AH133 S. aureus 0.21115 Extract by Cement Plant)/Ethyl acetate extract/50% 0.3435 Berberine -0.0074 50ug/ml ext + ber Hexane:ethyl acetate VLC Fraction. This extract 0.23455 25ug/ml 0.30935 12.5ug/ml exhibited slight activity. As little extract remained, this 0.3738 6.25ug/ml 0.31275 Cells fraction was not pursued. -0.0006 MHB
41
AH203 – N014795 Pouteria pallida/ Silica AH203 S. aureus 0.17105 Extract Liquid Chromatography Fraction. This extract 0.1746 Berberine -0.0017 50ug/ml ext + ber exhibited slight activity. As little extract remained, and 0.1514 25ug/ml 0.17345 12.5ug/ml fraction AH207 from this extract demonstrated 0.1788 6.25ug/ml 0.21305 Cells excellent activity, this fraction was not pursued. -0.0008 MHB
AH207 – N014795 Pouteria pallida/ Silica AH207 S. aureus 0.19955 Extract Liquid Chromatography Fraction. This extract 0.2445 Berberine -0.00765 50ug/ml ext + ber demonstrated the highest possible activity, inhibiting S. -0.0071 25ug/ml -0.0022 12.5ug/ml aureus growth at all concentrations. This fraction was 0.0534 6.25ug/ml 0.21455 Cells further fractionated by preparatory thin layer -0.00085 MHB chromatography, to yield fractions AH221 through AH228. These fractions were submitted for activity testing. None of the subsequent fractions exhibited activity.
AH209 – N014795 Pouteria pallida/ Silica AH209 S. aureus 0.1958 Extract Liquid Chromatography Fraction. This extract 0.1859 Berberine 0.0325 50ug/ml ext + ber exhibited slight activity. As little extract remained, and 0.15085 25ug/ml 0.1533 12.5ug/ml fraction AH207 from this extract demonstrated 0.18295 6.25ug/ml 0.2196 Cells excellent activity, this fraction was not pursued. -0.00095 MHB
AH235 – N102279 Thuja occidentalis/Silica AH 235 S. aureus 0.19425 Extract Liquid Chromatography Fraction. This fraction 0.2052 Berberine 0.0456 50ug/ml ext + ber exhibited moderately good activity. As the fraction 0.1011 25ug/ml 0.13445 12.5ug/ml appeared visually to be relatively pure, the elucidation 0.1434 6.25ug/ml 0.2167 Cells of a molecular structure was attempted. Dr. Ramadas -0.00325 MHB
42
Sathunuru analyzed this extract by H1-NMR, C13-NMR, elemental analysis, high resolution mass spectrometry, and exhaustive acetylation. These results are submitted in Appendix D. Dr. Sathunuru proposed the following structure:
CH OH HO 3
HO COOH HO OH
Figure 4. Molecular Structure - 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid
A systematic name for this compound is 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid.
AH242 – N102279 Thuja occidentalis/Silica AH 242 S. aureus 0.1906 Extract Liquid Chromatography Fraction. This fraction 0.218 Berberine 0.0852 50ug/ml ext + ber exhibited slight MDRP-inhibition activity. Since the 0.1617 25ug/ml 0.18745 12.5ug/ml activity was very slight, and the extract was tarry and 0.2057 6.25ug/ml 0.1963 Cells complex, further fractionation is not being attempted at -0.0006 MHB this time.
43
AH245 – N102249 Thuja occidentalis/Ethyl AH245 S. aureus 0.107 Extract acetate VLC Fraction. 0.315 Berberine -0.001 50ug/ml ext + ber This fraction (1.42g) exhibited moderate activity, and 0.114 25ug/ml 0.23 12.5ug/ml some direct activity. This fraction is a potential 0.27 6.25ug/ml 0.323 Cells candidate for further fractionation and testing. -0.001 MHB
AH250 – N102791 Rhus trilobata/80% AH250 S. aureus 0.208 Extract Hexane:ethyl acetate VLC Fraction. 0.299 Berberine 0.02 50ug/ml ext + ber This fraction (230mg) exhibited good activity. -0.004 25ug/ml 0.023 12.5ug/ml Therefore, the extract was further fractionated by silica 0.217 6.25ug/ml 0.257 Cells liquid chromatography, yielding samples AH261 -0.003 MHB through AH269. None of these extracts exhibited MDRP inhibition activity. Numerical results for these extracts are pending.
AH258 – N014045 Gunnera AH258 S. aureus 0.344 Extract macrophylla/Ethyl acetate VLC Fraction. 0.314 Berberine 0.09 50ug/ml ext + ber This fraction (19mg) exhibited slight activity. Since the 0.136 25ug/ml 0.162 12.5ug/ml sample was expended in activity testing, and the activity 0.231 6.25ug/ml 0.35 Cells was slight, this sample is not being pursued. 0 MHB
44
Chapter V
CONCLUSION
This thesis provides a great deal of detail, and a great deal of data. All in all, one extract was isolated which showed multi-drug resistance pump inhibition on multiple extractions. The structure of a potential inhibitor molecule was proposed, based on spectroscopic analysis by nuclear magnetic resonance and high resolution mass spectroscopy. In this following section, the total of the results will be further reviewed, and ways of increasing the efficiency of future investigations will be discussed.
Extraction Technique. The original plant samples were extracted sequentially with hexane, ethyl acetate, and methanol. The reasoning here is to isolate first the non- polar substituents, then the moderately polar substituents, and finally the most polar ones. This scheme is not based on a prediction of the polarity of the target compounds, but rather to isolate, separately, all of the molecules that are small enough to dissolve in some solvent. Among the seventy results returned for plant extracts, nine positive results were reported. Five of these results were from hexane extracts and vacuum liquid chromatographic fractions of hexane extracts. All of these extracts showed good activity. Three positive results were from VLC fractions of ethyl acetate extracts. Of
45
these, one extract exhibited good activity, and the others exhibited slight activity. One good positive result was obtained from a VLC fraction of a methanol extract.
These results indicate that generally the hexane extracts are more fruitful. Good scientific practice indicates that all possible extracts should be pursued, but these data indicate that the first work should be completed on the first, hexane extract. This way, more positive results are likely to be obtained in a given timeframe.
Fractionation Technique. Plant extracts, obtained from raw plants or from outside sources, were then fractionated by vacuum liquid chromatography (VLC), flash liquid chromatography (flash LC) and preparative thin layer chromatography (prep
TLC.) Of these techniques, prep TLC is specifically for very small amounts of material, and is therefore the only choice under those circumstances.
For the other techniques, VLC quickly divides the extract into five fractions, providing extracts for testing. Flash LC provides fractions that are demonstrated to be relatively pure. That is, fractions which contain a small number of compounds, hopefully one. Unpromising fractions, which show no peaks by TLC either under UV or developed with ethanolic sulfuric acid, are discarded. Out of sixty-two VLC results returned, there were ten active fractions, for a success rate of 16%. Out of forty-two flash LC results returned, there were five active fractions, for a success rate of 9%.
Intuitively, this is a surprising result. It could be argued that the more rigorous flask LC technique should yield the best results, and the lower number of inactive fractions, because of the discarding of the blank ones. However, the VLC technique, which is much more rapid and uses less solvents and simpler apparatus, yielded slightly
46
more positive results. Therefore, these data indicate that, for generation of positive activity results, VLC is the superior technique.
Characterization of Active Fragments. In four cases, fractions that demonstrated good activity were further fractionated to identify the actual MDRP inhibitor molecule. In the case of Eriogonum brevicaule Nuttall fraction AH64 and
Pouteria pallida fraction AH207, prep TLC was employed. Results for AH64 are pending, but sample AH207 yielded no active fractions. For Eriogonum brevicaule
Nuttall fractions AH73, AH103, and AH 127, which were found to be similar in composition and combined, and Rhus trilobata fraction AH250, flash LC was used for further fractionation. The Eriogonum brevicaule Nuttall yielded one fraction of very slight activity, which was not pursued. The Rhus trilobata fractions exhibited no activity at all.
This seems to be the weak point in these procedures. With these procedures, the most promising extracts are consumed and the activity is lost. This leaves the investigator with no useful results. This procedure can be very much improved by finding a better way to fractionate and characterize the active fractions. Currently, high pressure liquid chromatography/mass spectroscopy (LC/MS) and chiral column high pressure liquid chromatography are being considered for this task. With these state-of- the-art techniques, more useful results may be obtained.
47
APPENDIX A
Figure 5. Molecular Structures of Certain Antibiotics
O H N H C CH S 3 3 O H C 3 OH N OH HO CH3 O CH3 H3C O H3C O OH O O penicillin G H O HO N(CH ) O 3 2 CH3 H O CH CH N(CH ) 3 HO 3H H 3 2 OH H C OH 3 OMe
NH2 OH erythromycin OH O OH O O
tetracycline
HO CH3 H3C OH NH CH3 H3C H O Cl N H N 2 CH3 O O H O O HO O O O O HO NH NH O 2 HO H Cl HN O O H H HO H OH OH O N H O OH OH
vancomycin 48
APPENDIX B
Extraction Log
This log summarizes the extracts prepared for this study, including the species extracted, identification of the fraction, and results obtained from the multi-drug resistance pump inhibition testing.
Extract Amount Plant Fraction Hits Name AH 1 0.63 g Eriogonum hexane - unfractionated No activity AH 2 0.04 g Eriogonum hexane/ 100%hexane Consumed - re-extract AH 3 0.1 g Eriogonum hexane/ 50%hexane-EA Direct activity AH 4 0.003 g Eriogonum none XXX AH 5 0.014 g Eriogonum hexane/ 50%EA-MeOH Direct activity AH 6 0.007 g Eriogonum hexane/ 100%MeOH Consumed - re-extract AH 7 0.3 g Eriogonum ethyl acetate - unfractionated Direct activity AH 8 0.0008 g Eriogonum none XXX AH 9 0.038 g Eriogonum ethyl acetate/ 50%hexane-EA Direct activity AH 10 0.004 g Eriogonum ethyl acetate/ 100%EA Direct activity AH 11 0.027 g Eriogonum ethyl acetate/ 50%EA-MeOH No activity AH 12 0.003 g Eriogonum ethyl acetate/ 100%MeOH Consumed - re-extract AH 13 6.2 g Eriogonum ethanol - unfractionated Direct activity AH 14 0.001 g Eriogonum ethanol/ 100%hexane Direct activity AH 15 0.011 g Eriogonum ethanol/ 50%hexane-EA Direct activity AH 16 0.039 g Eriogonum ethanol/ 100%EA Direct activity AH 17 0.794 g Eriogonum ethanol/ 50%EA-MeOH Direct activity AH 18 0.329 g Eriogonum ethanol/ 100%MeOH Direct activity AH 19 0.066 g Eriogonum hexane/ 100%hexane no results returned AH 20 0.066 g Eriogonum hexane/ 100%hexane/ silica gel No activity AH 21 0.048 g Eriogonum ethyl acetate/ 100%MeOH No activity AH 22 0.048 g Eriogonum ethyl acetate/ 100%MeOH/ silica gel No activity AH 23 1.34 g Goldenseal hexane - unfractionated no results returned AH 24 0.027 g Goldenseal none XXX AH 25 0.26 g Goldenseal hexane/ 50%hexane-EA no results returned AH 26 0.034 g Goldenseal hexane/ 100%EA no results returned AH 27 0.064 g Goldenseal hexane/ 50%EA-MeOH no results returned AH 28 0.02 g Goldenseal none XXX AH 29 3.21 g Goldenseal ethyl acetate - unfractionated no results returned AH 30 0.027 g Goldenseal none XXX
49
Appendix B. Extraction Log (continued)
Extract Amount Plant Fraction Hits Name AH 31 0.56 g Goldenseal ethyl acetate/ 50%hexane-EA no results returned AH 32 0.158 g Goldenseal ethyl acetate/ 100%EA no results returned AH 33 0.528 g Goldenseal ethyl acetate/ 50%EA-MeOH no results returned AH 34 0.074 g Goldenseal ethyl acetate/ 100%MeOH no results returned AH 35 14.8 g Goldenseal methanol - unfractionated no results returned AH 36 0.063 g Goldenseal methanol/ 100%hexane no results returned AH 37 0.25 g Goldenseal methanol/ 50%hexane-EA no results returned AH 38 0.096 g Goldenseal methanol/ 100%EA no results returned AH 39 1.62 g Goldenseal methanol/ 50%EA-MeOH no results returned AH 40 2.09 g Goldenseal methanol/ 100%MeOH no results returned AH 41 0.51 g Biehl #45 methanol - unfractionated no results returned AH 42 0.009 g Biehl #45 none XXX AH 43 0.042 g Biehl #45 methanol/ 50%hexane-EA no results returned AH 44 0.021 g Biehl #45 methanol/ 100%EA no results returned AH 45 0.11 g Biehl #45 methanol/ 50%EA-MeOH no results returned AH 46 0.1 g Biehl #45 methanol/ 100%MeOH no results returned AH 47 0.39 g Biehl #246 ethyl acetate - unfractionated no results returned AH 48 0.026 g Biehl #246 ethyl acetate/ 100%hexane no results returned AH 49 0.16 g Biehl #246 ethyl acetate/ 50%hexane-EA no results returned AH 50 0.047 g Biehl #246 ethyl acetate/ 100%EA no results returned AH 51 0.15 g Biehl #246 ethyl acetate/ 50%EA-MeOH no results returned AH 52 0.018 g Biehl #246 ethyl acetate/ 100%MeOH no results returned AH 53 Eriogonum hexane - unfractionated no results returned AH 54 0.25 g Eriogonum hexane/ 100%hexane No activity AH 55 1.16 g Eriogonum hexane/ 50%hexane-EA no results returned AH 56 0.075 g Eriogonum hexane/ 100%EA no results returned AH 57 0.042 g Eriogonum hexane/ 50%EA-MeOH no results returned AH 58 0.3 g Eriogonum hexane/ 100%MeOH No activity AH 59 2.89 g Eriogonum ethyl acetate - unfractionated no results returned AH 60 0.04 g Eriogonum ethyl acetate/ 100%hexane no results returned AH 61 0.56 g Eriogonum ethyl acetate/ 50%hexane-EA no results returned AH 62 0.23 g Eriogonum ethyl acetate/ 100%EA no results returned AH 63 0.33 g Eriogonum ethyl acetate/ 50%EA-MeOH no results returned AH 64 0.099 g Eriogonum ethyl acetate/ 100%MeOH TLC – AH270-AH274 AH 65 10.4 g Eriogonum methanol - unfractionated no results returned AH 66 0.003 g Eriogonum none XXX AH 67 0.29 g Eriogonum methanol/ 50%hexane-EA no results returned AH 68 0.26 g Eriogonum methanol/ 100%EA no results returned AH 69 6.14 g Eriogonum methanol/ 50%EA-MeOH no results returned AH 70 0.8 g Eriogonum methanol/ 100%MeOH no results returned AH 71 0.94 g Hillside hexane - unfractionated Slight activity - Check fractions AH 72 0.048 g Hillside hexane/ 100%hexane No activity AH 73 0.56 g Hillside hexane/ 50%hexane-EA Direct activity AH 74 0.042 g Hillside hexane/ 100%EA No activity AH 75 0.34 g Hillside hexane/ 50%EA-MeOH No activity AH 76 0.049 g Hillside hexane/ 100%MeOH No activity AH 77 1.38 g Hillside ethyl acetate - unfractionated No activity AH 78 0.017 g Hillside none XXX AH 79 0.1 g Hillside ethyl acetate/ 50%hexane-EA Direct activity AH 80 0.14 g Hillside ethyl acetate/ 100%EA Direct activity 50
Appendix B. Extraction Log (continued)
Extract Amount Plant Fraction Hits Name AH 81 0.45 g Hillside ethyl acetate/ 50%EA-MeOH Slight activity - not persued AH 82 0.078 g Hillside ethyl acetate/ 100%MeOH No activity AH 83 15.6 g Hillside methanol - unfractionated Direct activity AH 84 0.002 g Hillside none XXX AH 85 0.092 g Hillside methanol/ 50%hexane-EA Direct activity AH 86 0.12 g Hillside methanol/ 100%EA Direct activity AH 87 0.716 g Hillside methanol/ 50%EA-MeOH Direct activity AH 88 1.36 g Hillside methanol/ 100%MeOH Direct activity AH 89 0.94 g Leaves hexane - unfractionated No activity AH 90 0.009 g Leaves hexane/ 100%hexane No activity AH 91 1.47 g Leaves hexane/ 50%hexane-EA No activity AH 92 0.023 g Leaves hexane/ 100%EA No activity AH 93 0.02 g Leaves hexane/ 50%EA-MeOH No activity AH 94 0.016 g Leaves hexane/ 100%MeOH No activity AH 95 2 g Leaves ethyl acetate - unfractionated No activity AH 96 0.004 g Leaves none XXX AH 97 2.4 g Leaves ethyl acetate/ 50%hexane-EA No activity AH 98 0.05 g Leaves ethyl acetate/ 100%EA No activity AH 99 0.408 g Leaves ethyl acetate/ 50%EA-MeOH No activity AH 100 1.2 g Leaves ethyl acetate/ 100%MeOH No activity AH 101 7.55 g Leaves methanol - unfractionated No activity AH 102 0.004 g Leaves none XXX AH 103 0.081 g Leaves methanol/ 50%hexane-EA Combined with AH127 AH 104 0.036 g Leaves methanol/ 100%EA No activity AH 105 2.51 g Leaves methanol/ 50%EA-MeOH No activity AH 106 2.72 g Leaves methanol/ 100%MeOH No activity AH 107 0.18 g Roots hexane - unfractionated Slight activity - Check fractions AH109-AH112 AH 108 0.005 g Roots none XXX AH 109 0.046 g Roots hexane/ 50%hexane-EA no results returned AH 110 0.011 g Roots hexane/ 100%EA no results returned AH 111 0.26 g Roots hexane/ 50%EA-MeOH no results returned AH 112 0.2 g Roots hexane/ 100%MeOH no results returned AH 113 0.29 g Roots ethyl acetate - unfractionated no results returned AH 114 0.004 g Roots none XXX AH 115 0.029 g Roots ethyl acetate/ 50%hexane-EA no results returned AH 116 0.015 g Roots ethyl acetate/ 100%EA no results returned AH 117 0.082 g Roots ethyl acetate/ 50%EA-MeOH no results returned AH 118 0.013 g Roots none XXX AH 119 1.14 g Roots methanol - unfractionated no results returned AH 120 0.015 g Roots none XXX AH 121 0.008 g Roots none XXX AH 122 0.003 g Roots none XXX AH 123 0.25 g Roots methanol/ 50%EA-MeOH no results returned AH 124 0.45 g Roots methanol/ 100%MeOH no results returned AH 125 FxCP hexane - unfractionated No activity AH 126 FxCP hexane/ 100%hexane No activity AH 127 0.8 g FxCP hexane/ 50%hexane-EA Combined with AH103 AH 128 FxCP hexane/ 100%EA No activity AH 129 FxCP hexane/ 50%EA-MeOH No activity 51
Appendix B. Extraction Log (continued)
Extract Amount Plant Fraction Hits Name AH 130 0.16 g FxCP hexane/ 100%MeOH No activity AH 131 FxCP ethyl acetate - unfractionated No activity AH 132 FxCP none XXX AH 133 FxCP ethyl acetate/ 50%hexane-EA Slight activity - not persued AH 134 FxCP ethyl acetate/ 100%EA No activity AH 135 FxCP ethyl acetate/ 50%EA-MeOH No activity AH 136 FxCP ethyl acetate/ 100%MeOH No activity AH 137 FxCP methanol - unfractionated no results returned AH 138 FxCP none XXX AH 139 FxCP none XXX AH 140 FxCP none XXX AH 141 0.34 g FxCP methanol/ 50%EA-MeOH no results returned AH 142 1.62 g FxCP methanol/ 100%MeOH no results returned
AH 201 N014795 AA 29-36 No activity AH 202 N014795 B 41-47 No activity AH 203 N014795 C2 48-60 Slight activity - not persued AH 204 N014795 C4 48-60 No activity AH 205 N014795 C5 48-60 No activity AH 206 N014795 D 105-126 No activity AH 207 N014795 E 127-135 Fractionated by TLC - AH221-AH228 AH 208 N014795 F 136-150 No activity AH 209 N014795 G 151-163 Slight activity - not persued AH 210 N014795 H 164-174 consumed AH 211 AH73/103/127 111-115 No activity AH 212 AH73/103/127 121-125 No activity AH 213 AH73/103/127 149-157 No activity AH 214 AH73/103/127 158-165 No activity AH 215 AH73/103/127 188-195 No activity AH 216 AH73/103/127 196-217 No activity AH 217 AH73/103/127 234-245 No activity AH 218 AH73/103/127 246-273 Slight activity - not persued AH 219 AH73/103/127 299-308 No activity AH 220 AH73/103/127 326-340 No activity AH 221 N014795-AH207 Fraction #1 No activity AH 222 N014795-AH207 Fraction #3 No activity AH 223 N014795-AH207 Fraction #4 No activity AH 224 N014795-AH207 Fraction #1B No activity AH 225 N014795-AH207 Fraction #2B No activity AH 226 N014795-AH207 Fraction #3B No activity AH 227 N014795-AH207 Fraction #4B No activity AH 228 N014795 A No activity AH 229 N014795 I 175-204 No activity AH 230 N014795 J 205 No activity
52
Appendix B. Extraction Log (continued)
Extract Amount Plant Fraction Hits Name
AH 231 N014795 K 206-208 No activity AH 232 N014795 L 209-212 No activity AH 233 N014795 M 213-216 No activity AH 234 N102279 10-12A Direct activity AH 235 N102279 10-12B Structure obtained AH 236 N102279 13-29 No activity AH 237 N102279 30-32 No activity AH 238 N102279 33-36 No activity AH 239 N102279 37-40 No activity AH 240 N102279 43-44 No activity AH 241 N102279 45-63 No activity AH 242 N102279 64-76 Slight activity - not persued (complex fraction) AH 243 0.01 g N102249 None XXX AH 244 0.8 g N102249 50%hexane-EA No activity AH 245 1.42 g N102249 100%EA Slight activity AH 246 2.42 g N102249 50%EA-MeOH No activity AH 247 0.12 g N102249 100%MeOH no results returned AH 248 0.022 g N102791 None XXX AH 249 0.18 g N102791 80%hexane-EA 01 No activity AH 250 0.23 g N102791 80%hexane-EA 02 Active - fractionate AH 251 0.66 g N102791 100%EA No activity AH 252 3.02 g N102791 50%EA-MeOH no results returned AH 253 0.28 g N102791 100%MeOH No activity AH 254 0.012 g N014045 None XXX AH 255 0.1 g N014045 90%hexane-EA No activity AH 256 0.23 g N014045 50%hexane-EA No activity AH 257 0.89 g N014045 100%EA 01 No activity AH 258 0.019 g N014045 100%EA 02 Slight activity AH 259 4.48 g N014045 50%EA-MeOH No activity AH 260 0.2 g N014045 100%MeOH no results returned AH 261 N102791-AH250 21-52 No activity AH 262 N102791-AH250 53-68 No activity AH 263 N102791-AH250 73-96 No activity AH 264 N102791-AH250 102-110 No activity AH 265 N102791-AH250 116-121 No activity AH 266 N102791-AH250 124-137 No activity AH 267 N102791-AH250 138-151 No activity AH 268 N102791-AH250 152-155 No activity AH 269 N102791-AH250 163-168 No activity AH 270 0.005 g Eriogonum-AH64 TLC fraction – pink pending AH 271 0.005 g Eriogonum-AH64 TLC fraction – dark drown pending AH 272 0.005 g Eriogonum-AH64 TLC fraction – brown pending AH 273 0.004 g Eriogonum-AH64 TLC fraction – brown #2 pending AH 274 0.004 g Eriogonum-AH64 TLC fraction – dark brown #2 pending
53
APPENDIX C
Table C-1. MDRP Activity Results - Eriogonum
AH 1 AH 1 AH2 AH2 AH 3 AH 3 S. aureus 0.093 0.137 0.17 0.141 -0.131 -0.062 extract 0.193 0.146 0.167 0.171 0.174 0.162 berberine 0.084 0.135 0.041 0.035 -0.287 -0.163 500 ug/mL ext + ber 0.042 0.069 0.085 0.072 -0.097 -0.136 250 ug/mL 0.097 0.086 0.123 0.103 -0.027 -0.009 125 ug/mL 0.102 0.097 0.136 0.111 0.049 0.075 62.5 ug/mL 0.246 0.223 0.21 0.195 0.215 0.201 cells 0.006 0.001 0.001 0.000 0.002 -0.002 MHB
AH 5 AH 5 AH 6 AH 6 AH 7 AH 7 S. aureus 0.049 0.021 0.192 0.193 -0.208 0.036 extract 0.161 0.192 0.206 0.203 0.188 0.171 berberine 0.002 0.015 0.055 0.057 0.027 0.04 500 ug/mL ext + ber 0.029 0.057 0.066 0.111 0.081 0.034 250 ug/mL 0.078 0.086 0.102 0.167 0.069 0.05 125 ug/mL 0.199 0.177 0.139 0.214 0.078 0.061 62.5 ug/mL 0.213 0.259 0.258 0.326 0.255 0.218 cells -0.001 -0.001 0.000 0.001 0.004 0.000 MHB
AH 9 AH 9 AH 10 AH 10 AH 11 AH 11 S. aureus -0.029 -0.128 -0.04 0.158 0.257 0.205 extract 0.196 0.176 0.182 0.183 0.192 0.148 berberine -0.254 -0.281 0.19 0.149 0.166 0.054 500 ug/mL ext + ber -0.057 -0.073 0.089 0.081 0.197 0.138 250 ug/mL 0.03 0.027 0.036 0.035 0.143 0.147 125 ug/mL 0.085 0.066 0.049 0.045 0.188 0.191 62.5 ug/mL 0.197 0.199 0.195 0.197 0.237 0.181 cells 0.001 -0.001 0.000 0.001 0.007 0.007 MHB
AH 12 AH 12 AH 13 AH 13 AH 14 AH 14 S. aureus 0.225 0.181 0.067 0.054 0.035 0.014 extract 0.19 0.215 0.151 0.135 0.148 0.161 berberine 0.091 0.071 0.063 0.06 -0.211 -0.206 500 ug/mL ext + ber 0.101 0.087 0.123 0.085 -0.107 -0.115 250 ug/mL 0.09 0.136 0.132 0.095 0.004 0.008 125 ug/mL 0.14 0.24 0.202 0.136 0.119 0.117 62.5 ug/mL 0.202 0.226 0.187 0.166 0.174 0.164 cells 0.001 0.000 0.006 0.001 0.000 0.001 MHB 54
Table C-1. MDRP Activity Results – Eriogonum (continued)
AH 15 AH 15 AH 16 AH 16 AH 17 AH 17 S. aureus -0.194 -0.204 0.045 0.033 0.048 0.055 extract 0.16 0.16 0.206 0.188 0.196 0.184 berberine -0.215 -0.231 -0.018 -0.028 0.044 0.043 500 ug/mL ext + ber -0.105 -0.12 -0.077 -0.029 0.115 0.097 250 ug/mL -0.03 -0.009 0.016 -0.028 0.143 0.119 125 ug/mL 0.04 0.063 0.037 0.01 0.11 0.126 62.5 ug/mL 0.191 0.254 0.229 0.213 0.207 0.216 cells 0.008 -0.002 0.099 0.002 0.000 0.000 MHB
AH 18 AH 18 S. aureus 0.062 0.067 extract 0.189 0.18 berberine 0.132 0.128 500 ug/mL ext + ber 0.143 0.139 250 ug/mL 0.157 0.128 125 ug/mL 0.119 0.156 62.5 ug/mL 0.191 0.203 cells -0.001 0.000 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
55
Table C-2. MDRP Activity Results – Eriogonum Hillside
AH71 AH72 AH73 AH74 AH75 AH76 S. aureus 0.1235 0.2089 -0.0199 0.1601 0.23035 0.23625 Extract 0.21595 0.19705 0.20515 0.2015 0.20025 0.20635 Berberine 0.0089 0.1754 -0.0322 0.10985 0.1826 0.19 500ug/ml ext + ber 0.0096 0.17345 -0.02365 0.114 0.1839 0.20265 250ug/ml 0.1104 0.1986 -0.01035 0.14255 0.1879 0.2152 125ug/ml 0.1403 0.19525 0.15775 0.112 0.19275 0.2215 62.5ug/ml 0.2462 0.2369 0.2383 0.2157 0.21375 0.23965 Cells -0.00065 -0.0003 0.00155 0.00035 1.00E-04 0.0006 MHB
AH77 AH79 AH80 AH81 AH82 AH83 S. aureus 0.19445 0.0866 -0.1126 0.118 0.2014 0.01825 Extract 0.2457 0.2166 0.2202 0.2196 0.22045 0.23855 Berberine 0.10265 5.00E-05 -0.1236 0.0697 0.11725 0.0026 500ug/ml ext + ber 0.13165 0.13625 0.0769 0.1009 0.12425 0.1077 250ug/ml 0.19705 0.1775 0.1701 0.1698 0.1563 0.16185 125ug/ml 0.25665 0.217 0.2041 0.2085 0.14005 0.14665 62.5ug/ml 0.2582 0.231 0.22665 0.22105 0.22235 0.25665 Cells -0.00085 -0.0006 -0.0004 -0.00035 -5.00E-05 -0.00025 MHB
AH85 AH86 AH87 AH88 S. aureus 0.0353 0.04395 0.00795 0.05585 Extract 0.26 0.2316 0.225 0.23105 Berberine 0.0922 -0.04285 0.08265 0.14225 500ug/ml ext + ber 0.0414 0.081 0.2008 0.1855 250ug/ml 0.06395 0.12285 0.15085 0.1475 125ug/ml 0.1312 0.09505 0.13505 0.1656 62.5ug/ml 0.24715 0.23075 0.23215 0.2341 Cells -0.00105 -0.0009 -0.0006 -0.00095 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
56
Table C-3. MDRP Activity Results – Eriogonum Leaves
AH89 AH90 AH91 AH92 AH93 AH94 S. aureus 0.26655 0.3003 0.296 0.28285 0.3081 0.3151 Extract 0.2209 0.2218 0.2123 0.21145 0.2097 0.21305 Berberine 0.2174 0.218 0.206 0.18035 0.27495 0.21305 50ug/ml ext + ber 0.2166 0.21725 0.24155 0.24685 0.2336 0.27095 25ug/ml 0.21475 0.2105 0.2354 0.2557 0.23035 0.33935 12.5ug/ml 0.21245 0.2138 0.22345 0.22365 0.2062 0.2373 6.25ug/ml 0.2197 0.22485 0.23235 0.22445 0.21815 0.21965 Cells -0.0058 -0.01685 -0.0046 -0.00445 -0.0053 0.2268 MHB
AH95 AH97 AH98 AH99 AH100 AH101 S. aureus 0.27705 0.2494 0.25895 0.25155 0.2227 0.3498 Extract 0.22105 0.20875 0.205 0.2151 0.20195 0.21735 Berberine 0.2156 0.20305 0.1686 0.25025 0.22815 0.10125 50ug/ml ext + ber 0.22385 0.20975 0.21855 0.2508 0.2376 0.13505 25ug/ml 0.2022 0.2174 0.2472 0.24815 0.2204 0.23895 12.5ug/ml 0.2104 0.21035 0.2573 0.24605 0.23775 0.21525 6.25ug/ml 0.22815 0.24185 0.2327 0.22455 0.22305 0.22315 Cells -0.00565 -0.00085 -0.0008 -0.0009 -0.00065 -0.00085 MHB
AH103 AH104 AH105 AH106 S. aureus 0.11145 0.2361 0.2625 0.23105 Extract 0.2095 0.1763 0.2049 0.20635 Berberine 0.088 0.18215 0.16345 0.1461 50ug/ml ext + ber 0.1959 0.1531 0.1856 0.1951 25ug/ml 0.23325 0.21205 0.19135 0.22335 12.5ug/ml 0.2246 0.224 0.2241 0.2253 6.25ug/ml 0.22105 0.2165 0.21165 0.2115 Cells -0.00085 -0.00365 -0.0045 -0.00485 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
57
Table C-4. MDRP Activity Results – Eriogonum Roots
AH107 S. aureus 0.1621 Extract 0.14275 Berberine 0.0942 50ug/ml ext + ber 0.03305 25ug/ml 0.16225 12.5ug/ml 0.2283 6.25ug/ml 0.2103 Cells -0.0051 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
58
Table C-5. MDRP Activity Results – Eriogonum Field by Cement Plant (FxCP)
AH125 AH126 AH127 AH128 AH129 AH130 S. aureus 0.2048 0.20835 0.16255 0.24215 0.2147 0.4329 Extract 0.3006 0.27905 0.2986 0.29215 0.30735 0.36075 Berberine 0.13815 0.2561 -0.0141 0.21955 0.35705 0.3175 50ug/ml ext + ber 0.35115 0.4441 -0.0082 0.29375 0.34365 0.33905 25ug/ml 0.3043 0.2891 0.17985 0.341 0.3147 0.30175 12.5ug/ml 0.29275 0.31105 0.2512 0.354 0.30155 0.34825 6.25ug/ml 0.2595 0.26075 0.2675 0.2569 0.30585 0.33675 Cells -0.00025 0.0001 -1.00E-04 -0.0004 -0.00025 0.00315 MHB
AH131 AH133 AH134 AH135 AH136 S. aureus 0.2634 0.21115 0.372 0.30215 0.41685 Extract 0.3784 0.3435 0.3437 0.3346 0.35625 Berberine 0.14965 -0.0074 0.3277 0.31935 0.3057 50ug/ml ext + ber 0.35915 0.23455 0.41635 0.6141 0.33955 25ug/ml 0.30935 0.30935 0.2986 0.37765 0.3003 12.5ug/ml 0.31885 0.3738 0.2913 0.3417 0.34695 6.25ug/ml 0.32645 0.31275 0.32845 0.3064 0.35675 Cells -5.00E-05 -0.0006 -0.00035 0 0.0006 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
59
Table C-6. MDRP Activity Results – Eriogonum – AH73/AH103/AH127 Fractionation
AH211 AH212 AH213 AH214 AH215 AH216 S. aureus 0.2306 0.2086 0.23585 0.2435 0.1822 0.2245 Extract 0.19555 0.18415 0.1783 0.1924 0.20425 0.20605 Berberine 0.263 0.22875 0.2148 0.20185 0.1571 0.13705 50ug/ml ext + ber 0.2285 0.2443 0.2238 0.2034 0.2907 0.26145 25ug/ml 0.1963 0.2028 0.20825 0.20805 0.2197 0.2627 12.5ug/ml 0.1827 0.1867 0.18965 0.24685 0.20905 0.2367 6.25ug/ml 0.19205 0.1976 0.20765 0.224 0.22055 0.2096 Cells -0.0011 -0.0013 -0.001 -0.001 -0.0006 -0.0008 MHB
AH217 AH218 AH219 AH220 S. aureus 0.36565 0.3559 0.3846 0.2354 Extract 0.19225 0.2032 0.2083 0.21095 Berberine 0.24225 0.091 0.4551 0.3065 50ug/ml ext + ber 0.46835 0.21725 0.60485 0.2671 25ug/ml 0.33265 0.2079 0.34765 0.24805 12.5ug/ml 0.23425 0.2091 0.2515 0.21125 6.25ug/ml 0.2189 0.22255 0.2196 0.22105 Cells -0.00075 -0.00075 -0.0009 -0.00095 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
60
Table C-7. MDRP Activity Results – Northeastern Sample N014795 (Pouteria pallida)
AH201 AH202 S. aureus 0.2636 0.27245 Extract 0.15105 0.15345 Berberine 0.23455 0.26535 500ug/ml ext + ber 0.2025 0.26255 250ug/ml 0.19535 0.2674 125ug/ml 0.2443 0.2093 62.5ug/ml 0.18565 0.20495 Cells -0.03515 -0.0356 MHB
AH203 AH204 AH205 AH206 AH207 AH208 S. aureus 0.17105 0.24085 0.27645 0.2082 0.19955 0.24815 Extract 0.1746 0.17775 0.19135 0.20775 0.2445 0.1951 Berberine -0.0017 0.1842 0.18015 0.1402 -0.00765 0.23945 50ug/ml ext + ber 0.1514 0.1612 0.18195 0.16575 -0.0071 0.2121 25ug/ml 0.17345 0.1755 0.19505 0.2155 -0.0022 0.2369 12.5ug/ml 0.1788 0.16325 0.18195 0.20465 0.0534 0.19705 6.25ug/ml 0.21305 0.1982 0.20685 0.20875 0.21455 0.22145 Cells -0.0008 -0.0007 -0.00085 -0.001 -0.00085 -0.0013 MHB
AH209 AH210 S. aureus 0.1958 0.29415 Extract 0.1859 0.1776 Berberine 0.0325 -0.0019 50ug/ml ext + ber 0.15085 0.079 25ug/ml 0.1533 0.1757 12.5ug/ml 0.18295 0.1715 6.25ug/ml 0.2196 0.21305 Cells -0.00095 0.0004 MHB
61
Table C-7. MDRP Activity Results – Northeastern Sample N014795 (continued)
AH 221 AH 222 AH 223 AH 224 AH 225 AH 226 S. aureus 0.29595 0.23515 0.21805 0.2107 0.2528 0.27575 Extract 0.22305 0.183 0.17895 0.1867 0.17395 0.2085 Berberine 0.2854 0.14465 0.1853 0.1639 0.1884 0.2238 50ug/ml ext + ber 0.38295 0.1757 0.2051 0.16285 0.20795 0.2132 25ug/ml 0.20345 0.1955 0.1926 0.2606 0.19745 0.2204 12.5ug/ml 0.16185 0.2105 0.2029 0.17705 0.1832 0.20015 6.25ug/ml 0.24335 0.2203 0.23105 0.2083 0.2217 0.2318 Cells -0.0002 0.0002 -0.0005 -0.00035 -0.00015 -0.00035 MHB
AH 227 AH 228 AH 229 AH 230 AH 231 AH 232 S. aureus 0.3043 0.24145 0.301 0.2878 0.21845 0.2939 Extract 0.19985 0.1896 0.19025 0.1893 0.23245 0.2524 Berberine 0.1876 0.18485 0.10315 0.11415 0.1732 0.19885 50ug/ml ext + ber 0.24995 0.2027 0.21725 0.18895 0.20695 0.224 25ug/ml 0.22615 0.1787 0.14895 0.1922 0.1715 0.1995 12.5ug/ml 0.2236 0.18325 0.1742 0.16255 0.17545 0.1954 6.25ug/ml 0.24155 0.21305 0.19975 0.1947 0.20185 0.2225 Cells 0.00015 -0.0002 -0.0007 -0.0001 -0.00015 -0.0002 MHB
AH 233 S. aureus 0.24905 Extract 0.21465 Berberine 0.1969 50ug/ml ext + ber 0.24415 25ug/ml 0.20405 12.5ug/ml 0.2346 6.25ug/ml 0.26005 Cells -0.00015 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
62
Table C-8. MDRP Activity Results – Northeastern Sample N102279 (Thuja occidentalis)
AH 234 AH 235 AH 236 AH 237 AH 238 AH 239 S. aureus 0.0114 0.19425 0.1752 0.1952 0.2037 0.2511 Extract 0.2063 0.2052 0.20625 0.19325 0.19645 0.2179 Berberine 0.00985 0.0456 0.1548 0.1294 0.1688 0.2184 50ug/ml ext + ber 0.02455 0.1011 0.2268 0.2502 0.2345 0.27715 25ug/ml 0.07605 0.13445 0.20835 0.1895 0.19145 0.2112 12.5ug/ml 0.07455 0.1434 0.2163 0.1417 0.20295 0.22465 6.25ug/ml 0.2229 0.2167 0.21795 0.2158 0.2381 0.2462 Cells -0.00145 -0.00325 -0.00025 -0.00015 0 -0.0002 MHB
AH 240 AH 241 AH 242 S. aureus 0.222 0.1974 0.1906 Extract 0.20915 0.208 0.218 Berberine 0.1785 0.14755 0.0852 50ug/ml ext + ber 0.29395 0.2289 0.1617 25ug/ml 0.20115 0.19875 0.18745 12.5ug/ml 0.18575 0.20655 0.2057 6.25ug/ml 0.20985 0.2015 0.1963 Cells 0.0003 -0.0002 -0.0006 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
63
Table C-9. MDRP Activity Results – Northeastern Sample N102249 (Thuja occidentalis)
AH244 AH245 AH246 S. aureus 0.265 0.107 0.275 Extract 0.295 0.315 0.341 Berberine 0.173 -0.001 0.252 50ug/ml ext + ber 0.309 0.114 0.242 25ug/ml 0.334 0.23 0.254 12.5ug/ml 0.431 0.27 0.317 6.25ug/ml 0.301 0.323 0.33 Cells 0 -0.001 0 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
64
Table C-10. MDRP Activity Results – Northeastern Sample N102791 (Rhus trilobata)
AH249 AH250 AH251 AH253 S. aureus 0.269 0.208 0.251 0.22 Extract 0.287 0.299 0.356 0.344 Berberine 0.183 0.02 0.28 0.142 50ug/ml ext + ber 0.235 -0.004 0.248 0.19 25ug/ml 0.229 0.023 0.257 0.236 12.5ug/ml 0.294 0.217 0.287 0.252 6.25ug/ml 0.27 0.257 0.328 0.327 Cells 0 -0.003 0 -0.001 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
65
Table C-11. MDRP Activity Results – Northeastern Sample N014045 (Gunnera macrophylla)
AH255 AH256 AH257 AH258 AH259 S. aureus 0.257 0.454 0.182 0.344 0.232 Extract 0.294 0.316 0.276 0.314 0.321 Berberine 0.13 0.242 0.176 0.09 0.236 50ug/ml ext + ber 0.223 0.227 0.158 0.136 0.217 25ug/ml 0.184 0.231 0.236 0.162 0.214 12.5ug/ml 0.246 0.229 0.22 0.231 0.261 6.25ug/ml 0.277 0.309 0.274 0.35 0.28 Cells -0.001 -0.001 0 0 0 MHB
Legend 0.111 (italics) MDRP inhibition activity detected 0.049 (bold) Direct activity detected - not useful 0.195 No activity detected
66
Appendix D
STRUCTURE ELUCIDATION DATA FOR FRACTION AH235
Fraction AH235 – N102279 Thuja occidentalis exhibited good activity. As the
fraction appeared visually to be pure, the elucidation of the chemical composition was
pursued, and a molecular structure was proposed.
The high resolution mass spectroscopy data (Figure 8) showed a molecular ion at
m/z 412, indicating it to be an oleanolic acid33 derivative with no hydroxyls in the “C”
ring. (See structure on the following page.) It gave a positive Liebermann-Burchard
test,34 indicating the structure to be a terpenoid.35 The following structure, with a
molecular formula of C22H36O7, was proposed.
22 12 13 18 20 11 17 CH OH HO 3 16 1 9 14 15 2 10 8
3 7 4 5 6 HO COOH HO OH 21 19
Figure 6. Molecular Structure - 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid
67
1 The H NMR (400 MHz, CDCl3) (Figure 9) showed several hydroxyl groups.
Since hydroxyl groups are notoriously difficult to analyze by 1H NMR, the compound
was subjected to exhaustive acetylation with acetic anhydride in pyridine. This
procedure yielded the following structure:
CH OAc AcO 3 C D A B AcO COOH AcO OAc
Figure 7. Molecular Structure - 2α,3β,4α,19,22β-Pentahydroxy-21-oic acid acetate
This compound has the molecular formula C32H46O12, with an expected m/z
of 622.
1 The H NMR (400 MHz, CDCl3) (Figure 10) of the acetylated compound showed five acetate methyl signals at δ0.73, δ0.83, δ0.84, δ0.94 and δ1.20, and one quaternary
methyl group at δ1.57, in agreement with the oleane skeleton.36 The H-17 appeared at
δ2.40 as a complex multiplet. The five hydroxyls are assigned to C-2, C-3, C-4, C-19 and C-22 positions from their chemical shifts and signal pattern. The H-2 appeared at
δ2.44 as a multiplet, indicating that the C-2 hydroxyl group is α equatorial. H-3
appeared at δ4.32 as a broad doublet (J = 7.0Hz), indicating that the C-3 hydroxyl group
is β equatorial. There are several oleane terpenoids that have C-2 and C-3 di-equitorial
hydroxyls. Examples are sericic acid,37 tomentosic acid,38 and marsileagenin-A,39 which show similar signal patterns to those observed in this compound. H-17 of the acetate appeared as a broad singlet at δ5.74 indicating C-17 to be β axial. There are several C-17
68
β-hydroxylated olean terpenoids, such as protobasic acid,40 barringtonic acid,41 and
42 tanginol, where the H-17 appeared at δ5.74 as a broad singlet. The OCH2 protons at
C-22 of the acetate appeared as an AB quartet (δ1.87, δ2.06, J = 10.0Hz.) The OCH2 protons at C-19 merged with H-17 appeared as a multiplet at δ2.31.
Thus, from a study of 1H NMR and high resolution mass spectroscopy of this compound and its acetate, it is characteristic of 2α,3β,4α,19,22β-pentahydroxy-21-oic
acid. This compound may be called “occidentalic acid.”
69
Figure 8. High Resolution Mass Spectroscopy Analysis of Fraction AH235
70
Figure 9. Nuclear Magnetic Resonance Spectroscopy Analysis of Fraction AH235
71
Figure 10. Nuclear Magnetic Resonance Spectroscopy Analysis of Fraction AH235 after exhaustive acetylation.
72
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