Institut für Pharmazeutische Wissenschaften Bereich Pharmakognosie Karl Franzens Universität Graz
United States Department of Agriculture Agricultural Research Service Natural Products Utilization Research Unit University of Mississippi
Diplomarbeit
Bio guided isolation of phytotoxic coumarin derivatives of Thamnosma montana Torr. & Frém., a Rutaceae native to the southwestern United States
Zur Erlangung des akademischen Grades eines Magisters der Pharmazie an der Naturwissenschaftlichen Fakultät der Karl Franzens Universität Graz
vorgelegt von Aaron Sallegger Graz, Juni 2009 This thesis was composed between August 2008 and June 2009 at the United States Department of Agriculture Agricultural Research Service, Natural Products Utilization Research Unit, University of Mississippi and the Institute of Pharmaceutical Sciences Department of Pharmacognosy, University of Graz.
First of all, I want to thank Dr. Wolfgang Schühly for being a great and motivated supervisor. Without him, this project would not have taken place and I would never have had the chance to experience working in the United States of America.
I also want to thank Dr. Charles Cantrell for taking over supervision in Oxford and Dr. Stephen Duke for coordinating the bioassays. Dr. Cantrell was a great mentor and friend, always ready to guide me through this interesting project and help me to get set up in Oxford.
Furthermore, my thanks go to Amber Callahan Reichley, Solomon Green III and Bob Johnson for being the best technicians I ever had the honor to work with and for giving me an amazing time in Oxford. I will never forget you guys!
Very special thanks goes to my family for being with me all the time, emotionally, mentally and financially. Without them, I would not have been able to achieve my goals.
Moreover, I want to thank the “Büro für Internationale Beziehungen” and the “Naturwissenschaftliche Fakultät” for the generous funding.
I also wish to thank Rolf Muertter, Aaron Schusteff and “jrdnz” for granting me permission to use their beautiful pictures of Thamnosma montana , which gave my thesis a colorful touch.
Last but not least, I want to mention my numerous friends and all the people, who helped me along my way, making me the person I am. Thank y'all! Table of Contents
Table of Contents 1 Introduction...... 1 2 Literature review...... 2 2.1 Rutaceae...... 2 2.2 Thamnosma ...... 2 2.2.1 Classification...... 3 2.3 Thamnosma montana ...... 4 2.3.1 Botanical characteristics...... 4 2.3.2 Distribution...... 5 2.3.3 Ethnobotanical use...... 6 2.3.4 Constituents...... 7 2.4 Coumarins...... 7 2.4.1 Classification...... 8 2.4.1.1 Simple coumarins...... 8 2.4.1.2 Furanocoumarins...... 8 2.4.1.3 Pyranocoumarins...... 8 2.4.2 Biosynthesis...... 9 2.4.2.1 Simple coumarins...... 9 2.4.2.2 Furanocoumarins...... 9 2.4.3 Furanocoumarins and phototoxicity...... 10 2.5 Bioassays...... 10 2.5.1 Bioassay guided fractionation...... 11 3 Materials and methods...... 13 3.1 Chromatography...... 13 3.1.1. Analytical thin layer chromatography...... 13 3.1.2 Preparative TLC...... 13 3.1.3 Column chromatography...... 14 3.1.4 Analytical and preparative high performance liquid chromatography (HPLC)...... 14 3.1.5 GC MS...... 14 3.1.6 LC MS...... 15
I Table of Contents
3.2 NMR...... 15 3.3 Instrumentation...... 16 3.3.1 TLC and preparative TLC...... 16 3.3.2 Column chromatography...... 16 3.3.3 HPLC and preparative HPLC...... 16 3.3.4 GC MS...... 17 3.3.5 High resolution LC MS...... 17 3.3.6 NMR...... 17 3.4 Plant material...... 17 3.5 Extraction...... 18 3.6 Fractionation...... 18 3.7 Bioassays against A. stolonifera and L. sativa ...... 19 3.8 Bioassay guided fractionation...... 20 3.8.1 Fraction V6...... 21 3.8.2 Fraction V7...... 22 3.8.3 Fraction V7ppt...... 24 3.8.4 Fraction V8ppt...... 24 3.8.5 Fraction V9...... 24 3.8.6 Fraction V10...... 26 3.8.7 Fraction V11...... 28 3.9 Bioassay against Lemna paucicostata L...... 29 3.9.1 General...... 29 3.9.2 Conduction of L. paucicostata bioassay...... 30 4 Results and discussion...... 32 4.1 Structure elucidation...... 32 4.2 AS1WS37_V6_comp1 (Isoimperatorin)...... 33 4.3 AS1WS37_V6_comp2 (Suberosin)...... 35 4.4 AS1WS37_V7_B and AS1WS37_V7_ppt (Swietenocoumarin B)...... 38 4.5 AS1WS37_V8_ppt (Bergapten)...... 45 4.6 AS1WS37_V9_A_P2 (Psoralen)...... 48 4.7 AS1WS37_V9_C (Dehydrogeijerin)...... 50 4.8 AS1WS37_V10_C_cry (Isopimpinellin)...... 53
II Table of Contents
4.9 AS1WS37_V10_E_B1 (Epoxysuberosin)...... 55 4.10 AS1WS37_V10_E_B2 (Alloimperatorin methyl ether epoxide)...... 58 5 Summary...... 61 6 Appendix...... 63 6.1 Bibliography...... 63 6.2 Index of Figures...... 67 6.3 Index of Tables...... 68
III
Introduction
1 Introduction
Secondary metabolites from various higher plants are of high interest to both the biologist, who aimes to understand the reason for their production and the pharmacologist looking out for potential sources of substances useful for human beings either as pharmaceuticals, dietary supplements or pesticides. To date, out of the ten thousands of known natural products, many have found their way into an application in either medicine or agriculture.
The use of natural products as pesticides dates back to ancient Greece and to the Roman Empire. Humans always searched for methods to extinguish weeds, insects or other herbivores in order to optimize crop yields. Modern agricultural practices rely on chemicals for pest control, but concerns about a potential impact of these pesticides on the environment and the rise of organic farming places an emphasis on natural products. Nowadays, pesticides derived from natural sources represent a good alternative to their synthetic equivalents and are widely used in organic agriculture (Dayan et al., 2009).
The goal of this thesis was to isolate and determine the active compounds of a crude extract previously screened for phytotoxic, antifungal and algicidal activity. The dichloromethane (DCM) extract of Thamnosma montana Torr. & Frém. (Rutaceae) showed notable activity in inhibiting the growth of the dicot, Lactuca sativa L. (Asteraceae) and the monocot Agrostis stolonifera L. (Poaceae).
This thesis will provide information about the systematic examination of T. montana for biological activity, including details on the bioassay guided fractionation and the evaluation of the herbicidal activities of the isolated compounds.
1 Literature review 2 Literature review
2.1 Rutaceae
The Rutaceae or rue family, which is usually placed in the order Sapindales or Rutales, consists of about 155 genera totaling over 1600 species. It contains the economically important genus Citrus , which provides fruits (i.e. orange ( C. sinensis ), lemon ( C. x limon ), lime (various, mostly C. aurantifolia ) and grapefruit ( C. paradisi )), as well as essential oils used in perfumery. Moreover, it includes the notable species Pilocarpus jaborandi which provides pilocarpine for glaucoma treatment (Chase et al., 1999).
The genera of the family are mostly distributed in tropical and subtropical regions (see Figure 1) and comprise usually shrubs and trees with oil glands in leaves and other parts. The flowers are commonly in cymes with a superior ovary and no more than twice as many stamens as sepals or petals (usually 4 5 sepals or petals). There can be various types of fruits (Evans, 1999).
Figure 1: Distribution of the Rutaceae family 2.2 Thamnosma
The genus Thamnosma Torr. & Frém., also known as “desert rue” or “turpentine bush”, contains two species in the United States of America, T. texana and T. montana . They are found in the deserts and semideserts of the
2 Literature review southwestern United States, namely in California, Nevada, Arizona, Utah, New Mexico, Colorado and Texas (see Figure 2) (PLANTS, 2009).
Thamnosma is not only found on the North American continent. Two species are found in southwestern African deserts, one on Socotra and one species is distributed in Arabia and Somalia, showing an interesting disjunction of Thamnosma (Mabberley, 1997).
Figure 2: Distribution of Thamnosma in North America 2.2.1 Classification
Superdivision Spermatophyta – Seed plants Division Magnoliophyta – Flowering plants Class Magnoliopsida – Dicotyledons Subclass Rosidae Order Sapindales/Rutales Family Rutaceae – Rue familiy Genus Thamnosma Torr. & Frém. desert rue
3 Literature review
2.3 Thamnosma montana
2.3.1 Botanical characteristics
Thamnosma montana Torrey & Frémont, also called “turpentine broom”, “cordoncillo” or “mojave desert rue”, is a 30 80 cm tall shrub with a broom like stem of yellowish green color (see Figure 3). It is thickly covered with blisterlike glands and leafless most of the year. T. montana forms new branches from the root crown, leading to multiple stems arising from the ground. The purplish flower is bisexual with a four lobed, persistent calyx, four petals, eight stamens in two series and a deeply two lobed ovary with a thread like style (see Figure 4). T. montana flowers in spring, usually from February through May, depending on the location (it starts earlier in Arizona and blooms later in California). The leaves are very small and deciduous, they are shed soon after flowering. Reproduction is done by seed, the flower is animal pollinated as well as the fruits are dispersed by animals. The fruit is a capsule, deeply two lobed, containing one to three whitish seeds, opening at the tip (see Figure 6) (Kearney, 1960; Shreve, 1964).
Figure 3: Thamnosma Figure 4: Thamnosma montana - flower montana - whole plant
4 Literature review
Figure 6: Thamnosma montana - fruit
Figure 5: 1flowering branch with several dark, bluish-purple flowers, the leaves are on only the young lateral branch; 2branch with fruits; 3the curious bispheroidal fruit covered with minute glands Figure 7: Thamnosma montana - habitat
2.3.2 Distribution
T. montana is native to the Californian Mojave Desert south of Death Valley and Barstow. It is also found in Nevada's southern Nye and Clark counties and in southwestern Utah. Moreover, T. montana is distributed in Arizona, along the rivers Little Colorado and Colorado in the Grand Canyon region, as well as
5 Literature review in the Mojavean Desert and in the Arizona Desert going as far east as Gila and Pinal counties (see Figure 8). It grows on sunny, dry, rocky, or gravelly slopes and reaches elevations from 700 m up to 1600 m (Benson & Darrow, 1981, Kearney, 1960).
Figure 8: Distribution of T. montana
2.3.3 Ethnobotanical use
Native American tribes from the southwestern deserts used different preparations of T. montana in various ways. The Havasupai , for example, used a decoction of the leaves as an emetic or they rubbed pounded leaves onto the abdomen as painrelief. Also the use of a decoction as a laxative is reported.
Another tribe, the Kawaiisu , used decoctions of stems in both an analgetic way and as a cold remedy. Furthermore, crushed stems of the plant were used on open wounds to promote rapid healing. It is also reported that the powdered plant was used to cause men and horses to sweat. This preparation was used as a hunting medicine, as well. It was put on deer tracks to slow down the animals.
6 Literature review
The Shoshoni smoked dried stems together with tobacco for colds, whereas the Paiute used a decoction of the stems as a gynecological aid (Moerman, 1998).
An interesting described use is an infusion of the whole plant taken by medicine men “to go crazy like coyotes”. Regarding the statement of an old squaw, while being in this state, they were able to find things lost for a long time (Jaeger, 1941).
2.3.4 Constituents
All parts of T. montana were thoroughly investigated in the fifties (Bennet & Bonner, 1953), sixties (Dreyer, 1966; Kutney et al., 1969) and seventies (Kutney et al., 1970; Kutney et al., 1972; Chang et al., 1975) of the last century. During these investigations, some alkaloids and many coumarin derivatives were isolated and some of them showed growth inhibiting properties against tomato seedlings (Bennet & Bonner, 1953). Byakangelicin, isopimpinellin, alloimperatorin methyl ether and its 2'', 3'' diol, skimmianine and a dimeric coumarin, named thamnosin are just a few examples of the isolated substances (Kutney et al., 1972). The alkaloids are of no interest in this thesis, the focus will be laid on the coumarin derivatives and their phytotoxic properties.
2.4 Coumarins
Coumarins are phenylpropanoids arising from the phenylalanine metabolism via o coumaric acid and p coumaric acid, both cinnamic acid derivatives. About 1000 coumarin derivatives have been described and they are found in many different families, particularly in Fabaceae, Asteraceae as well as in Apiaceae and Rutaceae. In Rutaceae coumarins show their greatest diversity. The name is referred to “coumarou”, which is the vernacular name of Dipteryx odorata Willd., Fabaceae, from whose seeds coumarin itself was first isolated in 1820 (Bruneton, 1995).
7 Literature review
2.4.1 Classification
Coumarins are broadly categorized as follows: • Simple coumarins • Furanocoumarins • Pyranocoumarins
2.4.1.1 Simple coumarins
Simple coumarins are the hydroxylated, alkoxylated and alkylated derivatives of coumarin itself, along with their glycosides. Umbelliferone, herniarin and aesculetin are examples.
Alkylation often occurs as prenylation, either O prenylation or prenylation in 6 or 8 position of umbelliferone or herniarin, which leads in the latter case to suberosin. Alkylation also leads to more complex polycyclic coumarins, i. e. furano and pyranocoumarins (Brahmachari, 2009).
2.4.1.2 Furanocoumarins
Furanocoumarins have a five membered furan ring attached to the coumarin nucleus and occur as either linear (i.e. psoralen, bergapten) or angular (i.e. angelicin, visnadin) derivatives (Brahmachari, 2009).
Since most of the substances purified during this project are simple coumarins and furanocoumarins, the focus will be laid on these two subclasses.
2.4.1.3 Pyranocoumarins
These derivatives are similar to the furanocoumarins, except for the ring attached to the nucleus, which is a six membered pyran ring. There are four types, the xanthyletin type, the alloxanthyletin type, the seselin type and the dihydroseselin type (Brahmachari, 2009).
8 Literature review
2.4.2 Biosynthesis
2.4.2.1 Simple coumarins
The biosynthesis of coumarins starts either from ortho- hydroxylation of cinnamic acid to 2 coumaric acid or the para- hydroxylation to 4 coumaric acid. The next step is a photocatalyzed isomerization, which manifests in a configuration change from trans ( E) to cis ( Z) in the side chain and is followed by a spontaneous lactonization. The results are coumarin derived from 2 coumaric acid and umbelliferone from 4 coumaric acid, respectively.
Coumarin itself has a scent of freshly mown hay and there is evidence that the plants contain the glycosides of 2 and 4 coumaric acids whereas coumarin is synthesized after tissue injury as a result of enzymic hydrolysis and lactonization (Dewick, 2002).
2.4.2.2 Furanocoumarins
As mentioned above, prenylation in position 6 or 8 of a 7 hydroxy coumarin leads to furanocoumarins. Derivatives of 6 prenylated coumarins yield the linear and such of 8 prenylated coumarins angular homologs, respectively.
Whereas it was postulated for many years that cyclization of 6 or 8 isoprenylcoumarin needs an epoxide as intermediate for a nucleophilic attack by the hydroxyl group (Bruneton, 1995), these epoxides have not been demonstrated in any enzymatic system so far investigated. It is believed that some direct oxidative cyclization mechanism must be involved.
The cyclization is catalysed by a cytochrome P450 dependent mono oxygenase with NADPH and molecular oxygen as cofactors. Another cytochrome P450 dependent mono oxygenase cleaves the hydroxyisopropyl fragment as acetone from marmesin yielding psoralen. The cleavage is believed to be initiated by a radical abstraction process without involving any hydroxylated intermediates.
Psoralen can be the originator of further substituted furanocoumarins, such as isopimpinellin, bergapten or isoimperatorin (Dewick, 2002).
9 Literature review
2.4.3 Furanocoumarins and phototoxicity
It has long been known that certain plant species of Rutaceae and Apiaceae have phototoxic properties. After contact with the plant and exposure to sunlight, an acute dermatitis with blisters and vesicles can occur. Usually this is followed by long lasting hyperpigmentation.
The constituents responsible for these symptoms are linear furanocoumarins, which are abundant in these families. Especially psoralen, bergapten (the 5 methoxy derivative of psoralen) and xanthotoxin (the 8 methoxy derivative of psoralen) should be mentioned. It is known that these linear furanocoumarins can intercalate into DNA and undergo cycloadditions with pyrimidine bases. This process explains their mutagenic and carcinogenic properties but not the mechanism of their phototoxicity (Bruneton, 1995).
Bergapten and xanthotoxin are used to treat psoriasis (a widespread autoimmune disorder characterized by proliferation of skin cells) and vitiligo (a chronic skin disorder that causes loss of pigment and appears as pale spots of skin). The treatment is called PUVA (psoralen + ultraviolet – A radiation) and the result is a reduction of the cell proliferation rate. However, one must note that this treatment requires adequate preparation. Since the skin overreacts under UV exposure, it is necessary to use sunscreen, sunglasses and protective clothing. Moreover, one must note that this treatment is not free of side effects like gastrointestinal disorders and accelerated aging of the crystalline lens of the eye. Long term therapy may even induce cancer (Bruneton, 1995; Dewick, 2002).
Bergapten is also used in external suntan preparations. Due to its ability to absorb the near UV, the formation of melanin pigments is stimluted (Dewick, 2002).
2.5 Bioassays
Bioassays are experiments that are used to reveal bioactive constituents in e.g. natural products. They are carried out on living organisms, and can be divided into various groups, depending on the used life form. These life forms
10 Literature review could be whole animals, isolated organs of vertebrates, lower organisms like fungi, bacteria or insects, cultured cells such as cancer cells or isolated subcellular systems e.g. enzymes or receptors.
Bioassays are very useful and can save a lot of time during an isolation process because only the bioactive fractions are subject of further investigations. They should be easy to perform and revealing the results within short time. Bioassays that are not costly and of high capacity allow high throughput screening. Usually a “primary bioassay screen”, is followed by a “secondary screen” for further exploration. These “secondary screens” involve more specific testing and tend to be slow and costly (Rahman/Choudhary/Thomsen, 2001).
2.5.1 Bioassay-guided fractionation
Rahman, Choudhary & Thomsen (2001) describe four approaches to bioassay guided natural product drug discovery research: • a single bioassy technique to search for a specific biological activity, such as growth inhibition • a battery of specific bioassay techniques, where each procedure is directed to discover a different type of activity • a single bioassay technique designed for detection of multiple activities. This could be a cytotoxicity bioassay, which then also can be used to predict various biological activities e.g. insecticidal, antimicrobial or antitumor activities • the combinatory use of different bioassays to detect specific as well as multiple activities. The choice depends always on the target and on the available information. For example, it may have been observed that certain plants are not attacked by insects, therefore it might be useful to screen those plants for insecticidal compounds. Rahman, Choudhary & Thomsen provide a typical flow diagram of a basic bioassay guided fractionation (see Figure 9):
11 Literature review
Figure 9: Flow chart of a typical bioassay-guided fractionation
12 Materials and methods 3 Materials and methods
3.1 Chromatography
Chromatography plays an important role in pharmacognosy. Almost every purification step that was done during this project was based on a chromatographic method.
Chromatographic separation is based on the principle that different molecules with different physical and chemical characteristics interact differently with a stationary phase and a mobile phase. It is used in both an analytical and a preparative way. There are various branches of chromatography, i.e. liquid solid chromatography, liquid liquid chromatography and gas solid chromatography (Robinson, 1987).
3.1.1 Analytical thin-layer chromatography
A very common chromatographic method is the thin layer chromatography (TLC). The sample is spotted on a plate made of glass or another inert material, that is usually either coated with silica gel or reversed phase silica gel. In the latter the SiOH groups are modified with long chain alcohols (C 18) to a non polar stationary phase. After placing the plate in a chamber with the mobile phase, which is usually a mixture of different solvents, the mobile phase is drawn up the plate by capillary action, thus separating the dotted samples regarding their polarity. The TLC plates are usually impregnated with a fluorescence detector that fluoresces at 254 nm. Compounds that are not fluorescent themselves can now be seen as dark spots on the fluorescing plate (Robinson, 1987).
3.1.2 Preparative TLC
The separation principle is the same as for analytical TLC. The stationary phase on the plate is thicker, therefore, more material can be applied. In this project, the compounds separated with this method were UV active, so
13 Materials and methods detection was done using a UV lamp detecting at 254 nm and 366 nm. The spots of interest are marked with a pencil then carefully removed from the plate, suspended in solvent, extracted, filtered and dried (Robinson, 1987).
3.1.3 Column chromatography
For column chromatography glass columns of different sizes and diameters are packed with the stationary phase. The dissolved sample is placed onto the substrate at the top, as well as the mobile phase is applied from the top. Either gravity or a pump let the solvents go through the column, thus separating the sample with regard to their interaction between the stationary and the mobile phase. The separated substances are collected as they drip off the end of the column (Robinson, 1987).
3.1.4 Analytical and preparative high-performance liquid chromatography (HPLC)
The high performance of this instrument lies in the fact that the solvent is pressed through a column packed with very small sized particles (3 10 m) by a pump with a pressure up to 400 bar. At the end of the column is a detector (usually a photometric detector like a diode array detector) that visualizes the results. HPLC is used in both analytical and preparative experiments. While for analytical HPLC 8 12 L are injected, for preparative HPLC an injection volume up to 1 mL is possible, depending on the column dimensions (Robinson, 1987).
3.1.5 Gas chromatography – mass spectrometry (GC-MS)
In gas chromatography the stationary phase is packed into a thin column and the mobile phase is an inert gas like helium (others can be nitrogen, argon or hydrogen). To provide a constant gas flow, pressure gauges and flow meters are used. The sample is vaporized after injection, therefore, only molecules stable at the adjusted temperature can be used.
14 Materials and methods
The instrument used in this project had a mass spectrometer attached to the end of the column. This instrument separates particles by their mass difference. The molecules have to be ionized, so they are bombarded by electrons. After this step they are accelerated in an electric field and enter into a homogeneous magnetic field in which they are deflected, depending on their mass. Finally, the particles hit a detector and there mass can be evaluated (Robinson, 1987). If a database is integrated possible structures can be shown through comparing the evaluated spectrogram with saved spectrograms in the database.
3.1.6 Liquid chromatography – mass spectrometry (LC-MS)
Liquid chromatography linked to a high resolution mass spectrometer is used to determine the exact mass, the molecular weight and the molecular formula of the investigated substance.
3.2 NMR
The most important technique to determine both purity and the structure of natural compounds, is NMR spectroscopy, which means nuclear magnetic resonance spectroscopy. The sample is aligned in a constant magnetic field which is perturbed by an alternating magnetic field. The spinning nuclei of the combined atoms in a molecule interact with electromagnetic radiofrequency radiation. The nuclei of the substance react differently regarding their electronic environment and therefore, their position in the molecule and so information about the chemical structure is obtained (Robinson, 1987).
Usually 1H and 13 C spectroscopy is used, the latter perturbs the carbon atoms in the molecule and delivers information about the number of carbons and their chemical shift, regarding their position within the molecule. 1H spectroscopy perturbs the hydrogens and provides information about the chemical shift, coupling neighbours and the abundance of protons.
To determine the actual structure two dimensional NMR techniques are performed, like COSY, DEPT, HSQC and HMBC spectra, which provide more
15 Materials and methods information about the coupling neighbours of either protons or carbons over more than one bond (Silverstein/Bassler/Morrill, 1991).
3.3 Instrumentation
3.3.1 TLC and preparative TLC
In this project, thin layer chromatography was performed using glass backed, 10x20 cm silica gel GF 250 microns plates – UNIPLATE TM (Analtech, Inc., Newark, DE) with an integrated 254 nm fluorescence indicator. After UV detection at 254 nm and 366 nm, the plates were sprayed with Godin reagent (vanillin/ethanol and sulfuric acid with subsequent heating) to visualize the results.
For reversed phase TLC glass backed, 10x20 cm RP 18 F 254s 250 m (Merck, Germany) TLC plates were used.
PrepTLC was performed using glass backed, 20x20 cm silica gel 60 F 254 0.5 mm P TLC plates (Merck, Germany).
3.3.2 Column chromatography
Column chromatography was performed using a Biotage SP1 (Charlottesville, VA), with spx software version 2.0. Biotage 40+M (40 63 m, 60 Å, 40x150 mm) columns were used at a flow rate of 40 mL/min.
3.3.3 HPLC and preparative HPLC
During this project HPLC method development work was performed using an Agilent 1100 system equipped with a quaternary pump, autosampler, diode array detector, and vacuum degasser. As solvents acetonitril (ACN), water 0.1% TFA (trifluoroacetic acid), and methanol (MeOH) were used.
Preparative HPLC purifications were performed using a Waters Delta Prep system (Milford, MA) equipped with a diode array detector a binary pump and a Zorbax SB C18, 21.2 x 250 mm, 7 m column.
16 Materials and methods
3.3.4 GC-MS
GC MS were obtained using a Varian CP 3800 GC coupled to a Varian Saturn 2000 MS/MS. The GC was equipped with a DB 5 fused silica capillary column (30 m x 0.25 mm, with film thickness of 0.25 m) and operated using method CLC_split_off_32minMSEI MS Only , under the following conditions: Injector temperature: 240 °C Column temperature: 60 280 °C at 8 °C/min then held at 280 °C for 5 minutes Carrier gas: helium Injection volume: 1 5 L MS mass range: 40 650 m/z MS ionaziation energy: 70 eV
3.3.5 High resolution LC-MS
High resolution mass spectra were obtained using an Agilent 1100 HPLC coupled to a JEOL AccuTOF (JMS T100LC) (Peabody, MA).
All isolated compounds were dissolved in MeOH and injected directly into a 0.3 mL/min stream of MeOH. 20 L of sample (approximately 0.1 mg/mL) was injected manually at 0.5 min while mass drift compensation standards (PEG – positive ion, and L tryptophan – negative ion) were injected at 1.5 min over the course of a 2 min run.
3.3.6 NMR
1 13 H and C NMR spectra were recorded using CDCl3 (deuterated chloroform) or
DMSO d 6 (deuterated dimethylsulfoxide) as solvents on a Varian ANOVA 400 MHz spectrometer (Palo Alto, CA) with TMS as standard. 13 C multiplicities were derived from 90° and 135° DEPT experiments.
3.4 Plant material
The investigated plant material of Thamnosma montana Torr. & Frém. was collected by Dr. Wolfgang Schühly in June 2001 near Littlefield, Mohave Co,
17 Materials and methods
AZ, USA, in open dry vegetation. A voucher specimen (WS 37) is deposited at the Pullen Herbarium at the University of Mississippi, USA.
3.5 Extraction
The aerial parts of the plant (1350 g) were finely ground and subsequently extracted 3 times with each 1.5 L of DCM. The DCM extracts were evaporated to yield 45 g of crude extract.
3.6 Fractionation
20 g of this extract were coated on 40 g of silica gel and applied onto an open column (200 g, silica 60, 40 63 m, Sorbent Technologies) and eluted using a gradient of ethyl acetate in n hexane (stepwise 0, 3, 5, 7, 10, 15, 25, 40% EtOAc in n hexane, each step 400 mL). Similar fractions were combined on basis of their TLC pattern. This process yielded 15 fractions out of which 1 11 were selected for bioassay screening.
Figure 10: Flow-chart of bioassay-guided fractionation
18 Materials and methods
3.7 Bioassays against A. stolonifera and L. sativa
The fractionation of the DCM extract was guided by bioassays against Agrostis stolonifera L. and Lactuca sativa L. These are miniaturized whole plant bioassays, representing monocotyledons and dicotyledons, respectively. The advantage of this bioassay lies in the easy germination and rapid growth of the used species. The growing medium is water, but poor solubility of many natural products requires preparation of a stock solution. Acetone is a suitable transfer solvent that has no physiological effects at concentrations of less than 10% v/v. Dimethylsulfoxide (DMSO) or other solvents can be used at concentrations lower than 1% v/v. Usually, two controls are included, in one control, a well is filled with water and in the other control the well is filled with water and the equivalent amount of transfer solvent without the test substance (Dayan et al., 2000).
The tests were performed in sterile non pyrogenic polystyrene 24 well cell culture plates (CoStar 3524, Corning Inc., NY), each containing a filter paper (Whatman Grade 1, 1.5 cm), and either five L. sativa seeds (Iceberg A Crisphead from Burpee Seeds) or 10 mg of A. stolonifera seeds (Turf Seed, Inc of Hubbard, Oregon). The tested fractions were dissolved in DCM or methanol, placed on the filterpaper and dried, prior to adding them on the well. After placing them on the well, distilled deionized (DDI) H 2O was added.
While the test wells were filled with 180 L of DDI H 2O plus 20 L sample mixture, the control wells were only filled with DDI H 2O and the same amount of transfer solvent used for the test wells. All plate preparation was done in a sterile environment to lessen chances of possible contamination. All wells were covered and sealed with parafilm. Incubation was performed at 26 °C in a Percival Scientific CU 36L5 incubator under continuous light conditions at 120 mol/m²/s. To qualitatively evaluate the phytotoxicity, the amount of germination of the seeds of each well was compared with the untreated controls after 7 days. Qualitative estimation of the phytotoxicity was evaluated by using a rating scale of 0 5, 0 meaning no effect and 5 meaning no growth or germination of the seeds. Each experiment was repeated in duplicate.
19 Materials and methods
24 well plate
T = test fractions (with e.g. L. sativa )
C = control with only water
CS = control with water and transfer solvent
Figure 11: Conduction of a bioassay
3.8 Bioassay guided fractionation
After combining the fractions based on TLC similarities, using UV detection and Godin reagent to visualize the results, the fractions V1 to V11 were screened for phytotoxic activity as growth inhibitors against Lactuca sativa L. (lettuce) and Agrostis stolonifera L. (bentgrass) (Table 1). Based on the results of those bioassays, the fractions V1 V5 were discarded.
The fractions V6 to V11 showed considerable activity and were decided to be further purified.
Ranking Sample ID Tested conc. Solvent used Day Lettuce Agrostis
AS1WS37V1 1 mg/mL DCM 7 0 0 AS1WS37V2 1 mg/mL DCM 7 0 0 AS1WS37V3 1 mg/mL DCM 7 0 0 AS1WS37V4 1 mg/mL DCM 7 0 0 AS1WS37V5 1 mg/mL DCM 7 0 0 AS1WS37V6 1 mg/mL DCM 7 1 4 AS1WS37V7 1 mg/mL DCM 7 2 3 AS1WS37V7ppt 1 mg/mL DCM 7 2 3 AS1WS37V8ppt 1 mg/mL DCM 7 1 4 AS1WS37V9 1 mg/mL DCM 7 3 4 AS1WS37V10 1 mg/mL DCM 7 3 4 AS1WS37V11 1 mg/mL DCM 7 1 4
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 1: Bioassay results after first VLC
20 Materials and methods
3.8.1 Fraction V6
TLC – and 1H NMR analysis showed that this fraction consisted of three compounds. Since all of them were UV active, thin layer chromatography was used to find a solvent system to separate the substances from each other.
After trying different solvent systems, 100% chloroform (CHCl 3) yielded the best results.
It was decided to purify fraction V6 by flash chromatography, using a silica gel column as the stationary phase and a hexane/CHCl 3 gradient solvent system as mobile phase at a flow rate of 40 mL/min. Flash chromatography was the standard separation method used for all fractions. Therefore, the steps described here are applicable to all other fractions. 400 mg of sample were dissolved in 2 mL of DCM, applied to the samplet and dried in the desiccator for one hour. A samplet is a small, silica gel filled cartridge specialized to be put on top of the used silica gel column. In the meantime the silica gel column was equilibrated (see Table 2). After the drying process the samplet was applied to the instrument and elution was performed.
Steps % Hexanes % Chloroform Volume
Equilibration 50 50 396mL Step 1 50 – 0 50 – 100 1600mL Step 2 0 100 600mL
Table 2: Solvent system for fraction V6 A total of 96 24 mL fractions were collected and checked by TLC. TLC analysis revealed that the compounds were not yet separated.
The next approach to purify the compounds from fraction V6 was preparative silica gel TLC. Since 100% chloroform delivered good results in analytical TLC, 100% chloroform was used as mobile phase. After two wash cycles with each 100% acetone, 20 mg of the sample dissolved in 0.5 mL of dichloromethane were applied to the plate. The TLC chamber was filled with 100 mL of chloroform and the prepTLC plate was developed for 1 ½ hours.
21 Materials and methods
After development of the plate, UV detection was used to check the results and four different compounds were detected. This method resulted in a good separation, allowing to proceed with each 40 mg of sample per prepTLC plate. The spots of interest were scratched off the plate, the substances were suspended and extracted in 5 10 mL of methanol, filtered and dried.
Four different compounds were obtained and subsequently analysed and tested against bentgrass and lettuce (Table 3). The GC MS analysis showed that compound 1 and 2 looked interesting while compound 3 had the same retention time and molecular weight as fraction V7, and compound 4 turned out to be of no interest.
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass AS1WS37V6comp1 1 mg/mL MeOH 7 0 1 AS1WS37V6comp2 1 mg/mL MeOH 7 1 3 AS1WS37V6comp3 1 mg/mL MeOH 7 1 2 AS1WS37V6comp4 1 mg/mL MeOH 7 0 2
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 3: Bioassay results of V6 subfractions
After repetition of prepTLC for ten times, 66 mg of AS1WS37V6comp1 and 73 mg of AS1WS37V6comp2 were isolated. The third compound on the plate did not yield a decent amount of pure substance and was therefore discarded.
3.8.2 Fraction V7
TLC patterns indicated that fraction V7 consisted of one major compound, equal to fraction V7ppt. Again chloroform turned out to lead to the best separation results. 500 mg of fraction V7 were dissolved in 2 mL of DCM, applied to the samplet and dried in the desiccator for one hour. After equilibrating the column, elution was performed (see Table 4).
22 Materials and methods
Steps Chloroform (%) Volume (mL)
Equilibration 100 396 Step 1 100 2405
Table 4: Solvent system for fraction V7 96 24 mL fractions were collected and combined on the basis of TLC similarities to yield 8 distinct subfractions (Table 5).
Fraction Testtube # Amount (mg)
A 16 2.0 B 17 25 102.0 C 26 31 130.0 D 32 36 33.0 E 36 44 11.0 F 45 55 24.0 G 56 67 10.0 H 68 75 3.0
Table 5: Subfractions of V7 Regarding the proton spectrum and GC MS analysis, subfraction B and C showed the same major compound, whereas all the other fractions contained traces of this compound but nothing else of interest. Therefore, subfractions B, C, D and F were tested against lettuce and bentgrass, which yielded the following results seen in Table 6.
23 Materials and methods
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V7B 1 mg/mL DCM 8 1 3 AS1WS37V7C 1 mg/mL DCM 8 1 3 AS1WS37V7D 1 mg/mL DCM 8 1 3 AS1WS37V7F 1 mg/mL DCM 8 1 2
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 6: Bioassay results of V7 subfractions
This process resulted in the isolation of 102 mg pure compound AS1WS37V7B.
3.8.3 Fraction V7ppt
Fraction V7ppt is a precipitate of fraction V7 and crystallized while drying during the first separation. NMR analysis confirmed the presence of a pure compound AS1WS37V7ppt, so no further purification steps had to be done.
3.8.4 Fraction V8ppt
Fraction V8ppt is another precipitate and turned out to be pure, as well – pure compound AS1WS37V8ppt.
3.8.5 Fraction V9
The proton NMR spectra led to the assumption of three major compounds in this fraction. After trying different solvent systems on TLC, a solvent mix of hexanes and diethylether turned out to yield the best separation.
570 mg of fraction V9 were dissolved in 2 mL of DCM, applied to the samplet and dried in the desiccator for one hour. The following gradient was used for the silica gel column chromatography (Table 7):
24 Materials and methods
Steps Hexanes (%) Diethylether (%) Volume (mL)
Equilibration 75 25 400 Step 1 75 25 25 75 1720 Step 2 25 75 580
Table 7: Solvent system for fraction V9
96 24 mL test tubes were collected and combined based on TLC similarities leading to 3 distinct subfractions (Table 8).
Fraction Testtube # Amount (mg)
A 35 43 35.0 B 44 54 80.0 C 55 66 25.0
Table 8: Subfractions of V9
This resulted in 25 mg of pure compound AS1WS37V9C.
GC MS and proton NMR spectra showed that subfraction A was a mixture of two compounds and subfraction B contained the compounds of A and C.
Before any further purification steps were undertaken, all subfractions were tested against lettuce and bentgrass (Table 9).
25 Materials and methods
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V9A 1.0 mg/mL DCM 6 4 5 AS1WS37V9A 0.1 mg/mL DCM 6 1 4 AS1WS37V9B 1.0 mg/mL DCM 6 4 5 AS1WS37V9B 0.1 mg/mL DCM 6 1 4 AS1WS37V9C 1.0 mg/mL DCM 6 3 4 AS1WS37V9C 0.1 mg/mL DCM 6 1 2
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 9: Bioassay results V9 subfractions
As could be seen in Table 9, the subfractions were also active at 0.1 mg/mL.
Subfraction A was subject of further purification using reversed phase TLC plates to find a suitable solvent system since normal phase silica did not yield satisfying results.
80% Methanol and 20% water turned out to yield good separation results. Subfraction V9A was subjected to reversed phase prepTLC. 4 mg/mL were applied to each plate, after two wash cycles using 100% methanol and developed within 40 min.
UV detection showed three different compounds, the first one turned out to be out of interest and was discarded, the second one yielded 7 mg of pure compound AS1WS37V9AP2 while the third one was identified as a substance previously purified. The spots of interest were scratched off the plate, the substances were suspended and extracted in 5 10 mL of methanol, filtered and dried.
3.8.6 Fraction V10
General analysis by GC MS and proton NMR showed three major and one minor compound in this fraction. The same flash chromatography method as
26 Materials and methods seen in Table 7 was used to purify the substances. 400 mg were dissolved in 2 mL of DCM, applied to the samplet and dried in the desiccator for one hour. 96 24 mL test tubes were collected and combined based on TLC similarities to yield six distinct fractions (Table 10).
Fraction Test tube # Amount (mg)
A 33 41 106.0 B 42 46 106.0 C 47 53 373.0 D 54 60 155.0 E 61 65 198.0 F 66 96 128.0
Table 10: Subfractions of V10
Pure compound AS1WS37V10Ccry crystallized while drying. The whole procedure was continued three times, for 1.6 g of fraction V10 were available. All subfractions were tested against lettuce and bentgrass (Table 11).
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V10A 1.0 mg/mL DCM 6 3 5 AS1WS37V10A 0.1 mg/mL DCM 6 2 4 AS1WS37V10B 1.0 mg/mL DCM 6 4 5 AS1WS37V10B 0.1 mg/mL DCM 6 3 5 AS1WS37V10C 1.0 mg/mL DCM 6 3 5 AS1WS37V10C 0.1 mg/mL DCM 6 2 4 AS1WS37V10D 1.0 mg/mL DCM 6 3 5 AS1WS37V10D 0.1 mg/mL DCM 6 2 3 AS1WS37V10E 1.0 mg/mL DCM 6 3 4 AS1WS37V10E 0.1 mg/mL DCM 6 1 1 AS1WS37V10F 1.0 mg/mL DCM 6 1 4 AS1WS37V10F 0.1 mg/mL DCM 6 0 0
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 11: Bioassay results V10 subfractions
27 Materials and methods
After GC MS as well as proton NMR analysis, subfractions B, D & F were discarded, while subfraction A turned out to be the same as an already purified compound.
Subfraction C was dissolved in ethyl acetate (EtOAc) and put into the freezer for recrystallization to yield 101 mg of pure compound AS1WS37V10Ccry.
Subfraction E was subject to further purification. The proton NMR analysis confirmed the existence of two compounds.
After TLC evaluation, these compounds were separated on silica gel flash chromatography, using the following gradient (Table 12):
Steps Hexanes (%) Acetone (%) Volume (mL)
Equilibration 100 0 400 Step 1 100 – 90 0 – 10 400 Step 2 90 10 600 Step 3 90 – 70 10 – 30 1150 Step 4 70 – 0 30 – 100 600
Table 12: Solvent system for subfraction V10E
130 24 mL test tubes were collected and combined on the basis of TLC similarities into four subfractions. This resulted in the isolation of the pure compounds AS1WS37V10EB1 (19 mg) and AS1WS37V10EB2 (120 mg).
GC MS and proton NMR analysis revealed that subsubfractions B3 and B4 were of no interest, so they were discarded.
3.8.7 Fraction V11
Fraction V11 turned out to be very difficult to separate. Although different methods were tried, none yielded decent results.
At first, silica gel flash chromatography was performed using the same solvents and gradient as seen in Table 7. 400 mg were dissolved in 2 mL of DCM, applied to the samplet and dried in the desiccator for one hour. After the
28 Materials and methods elution the TLC showed no decent separation. Furthermore, about 60% of the applied 400 mg were lost on the column.
The next trial was performing prepHPLC equipped with a reversed phase column using water 0.1% TFA/ACN as solvents. Prior to the separation, a method on the analytical HPLC was developed.
The final method details can be seen in Table 13:
Water (%) Time (min) ACN (%) + 0.1% TFA 0.00 30.0 70.0 10.00 30.0 70.0 30.00 50.0 50.0 35.00 100.0 0.0 40.00 100.0 0.0
Table 13: HPLC-method details V11
Upscaling of the injection volume for the prepHPLC was done in steps from 100 L, 300 L, 500 L, 700 L to 1 mL per injection. The separation was successful enough to proceed with an injection volume of 1 mL of a 10 mg/mL solution. 400 mg were dissolved in 40 mL of methanol and injected at a flow rate of 20 mL/min, UV detection was performed at 220 nm.
Five peaks were collected, the ACN was evaporated on the rotovapor, while the water was removed in the lyophilizer. This process yielded five subfractions, which either were not pure, regarding their proton NMR spectra, showed poor solubility or the amount was too small to perform further investigations.
3.9 Bioassay against Lemna paucicostata L.
3.9.1 General
Many species of the genus Lemna (duckweed) are commonly used in bioassays to evaluate phytotoxicity of substances. Lemna is a free floating monocotyledonous plant that grows very fast by clonal propagation, thus
29 Materials and methods offering genetically homogeneous material (Michel et al., 2004). It is distributed over the whole United States and Canada (PLANTS, 2009).
The fact that Lemna could easily be grown under sterile conditions in a nutrient solution turns this plant into the perfect candidate for miniaturized bioassays. Another fact that makes this genus suitable for growth tests is the two dimensional growth on the water surface, making it easy to evaluate the growth inhibition by monitoring the frond area. This is nondestructive and allows very accurate determination of growth in a dose response manner, also providing an IC 50 . Therefore, it is possible to evaluate growth inhibitory properties of tested compounds quantitatively, compared to the more qualitative evaluation of the lettuce and bentgrass bioassays.
In this bioassay the species L. paucicostata is used. It is smaller than L. minor and L. gibba , supporting miniaturization (Michel et al., 2004).
3.9.2 Conduction of L. paucicostata bioassay
All lemna were grown in Hoagland's No. 2 Basal Salt Mixture (Sigma H2395) (1.6 g/L) with added iron (1 mL of 1000X FeEDTA solution to 1 L of Hoagland media). The 1000X iron solution contained 18.355 g/L of Fe EDTA. The pH of the media was adjusted to 5.5 with 1 N NaOH. The media were filter sterilized using a 0.2 m filter and stored in sterile 1 L bottles. The lemna stocks were grown in approximately 100 mL of media in sterile baby food jars with vented lids. Lemna stocks were started from one or two three frond plants and grown in a Percival Scientific CU 36L5 incubator under continuous light conditions at 26°C and 120.1 mol/m²/s average light intensity. The medium was changed every 2 to 3 days or new stocks were prepared in fresh medium. Plant doubling time was approximately 24 hours.
The tests were conducted using non pyrogenic polystyrene sterile 6 well plates (CoStar 3506, Corning Inc., NY). Each well contained 4950 L of the Hoagland's media plus 50 L of water, or the solvent, or the compound dissolved in the appropriate solvent (at a concentration of 100x). The final concentration of the solvent was therefore approximately 1% by volume.
30 Materials and methods
When setting up the test, the solutions were prepared in 50 mL centrifuge tubes. 14850 L of Hoagland was pipetted into a tube and 150 L of the 100x concentration of the compound were added. Afterwards the tubes were vortexed and 5000 L of them were aliquoted into three wells. A graphic template of the 6 well plates was used to randomly put the triplicates in any of the plates. This allowed to rule out any differing light intensities in the growth chamber. The sheet was then used to set up the test template using the LemnaTec software (Würselen, Germany).
Each well was inoculated with two 3 frond plants of the same age (4 5 days old) and approximate size. All 6 well plates were incubated in a Percival incubator under the same conditions as described above. The plant areas were measured at day 0 and day 7 and different days in between. The software provided the frond number and the total frond area, as well as color classes to indicate any chlorotic or necrotic effects.
The obtained raw data was used in spreadsheets that calculate change in frond area over time. The averages of the triplicates were plotted along with the standard deviation using SigmaPlot software program. The graph shows the growth in percent versus the logarithm of concentration ( M).
31 Results and discussion
4 Results and discussion
4.1 Structure elucidation
The structure elucidation of all isolated compounds was performed following the same general manner. The first step was always a TLC evaluation to get an overview of the different fractions. Usually the same solvent system was used for TLC as for the actual separation process. The compounds were detected at 254 nm and 366 nm.
Further, a GC MS was run to check for impurities and to get an idea of the potential molecular weight of the isolated substance. Since the used instrument is linked to a database, it was possible to compare the evaluated spectra with spectra sets from the database to suggest probable structures.
The next step comprised the use of 1H NMR to check for purity and to obtain information about the class of molecule, characteristic groups etc. If the substance was pure regarding to the proton data, a 13 C NMR run was performed. The 13 C NMR revealed the number of carbons in the molecule.
At last, an LC MS run was done. Together with the information on the potential molecular weight and the number of carbons, it was generally possible to establish the molecular formula.
With all the spectra obtained information, literature was browsed for comparable NMR data. Since most of the isolated substances in this project are known coumarins, literature commonly offered NMR data that completely confirmed the structure of the isolated compounds. In one case DEPT, COSY, HSQC and HMBC runs were performed because the available literature data of the estimated structure was old and only 1H NMR data was recorded. In another case no 13 C data were published and so the structure could be confirmed by comparing published 1H NMR data after getting more detailed structural information via DEPT and COSY spectra.
32 Results and discussion
4.2 AS1WS37_V6_comp1 (Isoimperatorin)
4''
3''
2'' 5''
1'' O
5 4 2' 6 3 4a
1' 8a 2 7 O O O 8 1 Figure 12: Isoimperatorin
Molecular weight: 270.28
Molecular formula: C 16 H14 O4
Isoimperatorin (Figure 12) is a coumarin belonging to the linear furanocoumarin group and it is etherified with a prenyl group in position 5. It was discovered in T. montana before by Kutney et al. (1972). The recorded 1H and 13 C NMR spectra were in complete agreement with already published values (Table 14) (Masuda et al., 1998) and so the structure could be confirmed completely. The molecular weight and molecular formula were obtained by LC MS.
33 Results and discussion
δ 13 C (ppm) δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature 18.4 18.3 1.67 s, 3H 1.70 s, 3H 26.0 25.8 1.77 s, 3H 1.80 s, 3H 69.9 69.8 4.89 d, 2H 4.92 d, 2H 94.3 94.2 5.50 t like, 1H 5.54 t like, 1H 105.3 105.1 6.23 d, 1H 6.27 d, 1H 107.6 107.5 6.92 d, 1H 6.96 d, 1H 112.7 112.6 7.10 s, 1H 7.15 s, 1H 114.3 114.2 7.56 d, 1H 7.60 d, 1H 119.2 119.1 8.13 d, 1H 8.16 d, 1H 139.8 139.6 140.0 139.8 145.1 144.9 149.1 149.0 152.8 152.7 158.3 158.1 161.5 161.3
Table 14: NMR-data of isoimperatorin compared to Masuda et al. (1998)
Isoimperatorin (Table 16) did not show significant inhibition in the lettuce and bentgrass bioassay. Therefore, isoimperatorin could obviously not be responsible for the activity found in fraction V6.
The growth inhibitory effects of all isolated substances were evaluated quantitatively in a duckweed ( Lemna paucicostata ) bioassay. The compounds were tested at concentrations ranging from 1000 M to 0.1 M. After each day four and day seven, the Lemna growth (in percent on y axis) was evaluated at each concentration (in M on x axis). This was the case for all purified compounds and will not be described in the other chapters.
As seen in Figure 13, the parameters measured during the test phase of 7 days did not yield evaluable data. After 4 days the average increase of the surface area ranged from about 150% to 220% at all concentrations. Whereas after 7 days the data on the y axis was scattered trendless between 400% and almost 600%. The software was not able to evaluate an IC 50 with this data.
34 Results and discussion
Figure 13: Dose-response curve of isoimperatorin
4.3 AS1WS37_V6_comp2 (Suberosin)
11 9 5 4
6 3 4a 10
8a 2 7 H3CO O O 11 8 1 Figure 14: Suberosin
Molecular weight: 244.29
Molecular formula: C 15 H16 O3
The second isolated constituent of fraction V6 was suberosin (Figure 14). Suberosin is a simple coumarin with an umbelliferone backbone. The hydroxy
35 Results and discussion group in position 7 is methylated and a prenyl group is added in position 6. It was isolated from T. montana before (Kutney et al., 1972). The molecular formula and the molecular weight were obtained by LC MS. 1H and 13 C NMR data were recorded and compared with data from possible structures in the literature (Table 15), which led to complete confirmation of the present compound (El Shafae & Ibrahim, 2003).
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 161.75 161.48 C3 112.89 112.72 6.22 d, 1H 6.20 d, 1H C4 143.86 143.59 7.61 d, 1H 7.59 d, 1H C4a 127.64 127.44 C5 127.56 127.36 7.17 s, 1H 7.15 s, 1H C6 112.04 111.85 C7 154.62 154.44 C8 98.64 98.45 6.76 s, 1H 6.75 s, 1H C8a 160.80 160.61 C9 27.95 27.75 3.30 d, 2H 3.28 d, 2H C10 121.48 121.31 5.27 m, 1H 5.26 m, 1H C11 133.86 133.61 C11 Me 17.95 17.72 1.70 s, 3H 1.68 s, 3H C11 Me 26.02 25.78 1.76 s, 3H 1.74 s, 3H 7 OMe 56.04 55.82 3.89 s, 3H 3.87 s, 3H
Table 15: NMR-data (CDCl 3) of suberosin compared to El-Shafae & Ibrahim (2003) Suberosin was tested against lettuce and bentgrass (Table 16) and showed very low activity on dicot growth, while the growth inhibiting activity on monocots was moderate. Comparing these results with results from the whole fraction V6, this outcome was expected since the mixture of the two compounds was not very active against lettuce but inhibited the growth of bentgrass.
36 Results and discussion
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V6 1 mg/mL DCM 7 1 4
AS1WS37V6comp1 1 mg/mL MeOH 7 0 1 AS1WS37V6comp2 1 mg/mL MeOH 7 1 3
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 16: Lettuce and bentgrass bioassay results of fraction V6 and of the isolated compounds
As mentioned above suberosin showed significant activity in the qualitative bioassay, whereas in the quantitative bioassay (Figure 15) no reasonable growth inhibition was measured at low concentrations. The IC 50 probably could be found at concentrations higher than 1000 M, therefore suberosin is not active and no IC 50 could be evaluated.
Figure 15: Dose-response curve of suberosin
37 Results and discussion
4.4 AS1WS37_V7_B and AS1WS37_V7_ppt (Swietenocoumarin B)
OCH3
5 4 2' 6 3 4a
1' 8a 2 7 O 8 O O 14 1
1'' 2''
3'' 4'' 5'' Figure 16: Swietenocoumarin B
Molecular weight: 284.31
Molecular formula: C 17 H16 O4
NMR and TLC evaluation proved AS1WS37V7B and AS1WS37V7ppt to be identical.
Literature did not provide any more recent NMR data for a substance with this molecular weight and formula, further NMR experiments including 2D experiments, were performed (DEPT, COSY, HSQC & HMBC). It was assumed that the compound was another linear furanocoumarin, substituted with a methoxy group and a prenyl group. The obtained spectra were thoroughly investigated and the following conclusions were drawn: 1H NMR spectrum (Figure 17): • there were 16 protons in the molecule • two methyl groups were existent • the molecule contained a methoxy group • four protons were in the aromatic region, all were doublets, so, probably each two of them coupled to each other
38 Results and discussion
• one multiplet with two protons suggested the existence of a CH 2 group coupling to other protons • it further revealed the number of protons for each peak 13 C NMR spectrum (Figure 18): • there were 17 carbons in the molecule • three of the carbons appeared in high field • one carbon signal was found at around 60 ppm, probably the carbon belonging to the methoxy group • the rest of the carbon signals was found in the aromatic region DEPT spectra:
• five CH groups, one CH 2 group, three methyls and seven quaternary carbons were found in the molecule COSY spectrum (Figure 19): • the peak at 7.92 ppm (d, 1H) coupled with the peak at 6.32 ppm (d, 1H) • the peak at 7.63 ppm (d, 1H) coupled with the peak at 6.81 ppm (d, 1H), thus showing each two carbons being each one proton coupling to each other suggesting the presence of two separated olefinic spin systems. This supported the structure of a linear furanocoumarin and showing that it must be substituted in positions 5 and 8 • the peak at 3.65 ppm (d, 2H) coupled with the peak at 5.08 ppm (m, 1H), both probably belonging to a side chain HMBC spectrum (Figure 21): • the two methyls (1.66 ppm and 1.81 ppm) coupled to two carbons 122.2 ppm (CH) and at 133.0 ppm (C quaternary); respectively
• the proton peak at 3.65 ppm, which belongs to a CH 2 group coupled to carbons at 133.0 ppm and to the one at 122.2 ppm, thus suggesting the side chain being the expected prenyl group • the peak at 122.2 ppm also coupled to one of the 125 ppm peaks and the peak at 114.3 ppm, suggesting that the side chain is linked to the side of the molecule, where those carbons were situated
39 Results and discussion
• the methoxy group coupled to carbon peaks at 131.3 ppm and 144.1 ppm, proving the methoxy group to be attached at position 5 • investigation of the other peaks supported the theory of a linear furanocoumarin structure and led to the postulation of the molecule seen in Figure 16. HSQC spectrum (Figure 20):
Protons coupling to carbons 1.66 ppm (s, 3H) 25.7 ppm 1.81 ppm (s, 3H) 18.3 ppm 3.65 ppm (d, 2H) 28.2 ppm 4.19 ppm (s, 3H) 61.6 ppm 6.32 ppm (d, 1H) 114.0 ppm 6.81 ppm (d, 1H) 105.9 ppm 7.63 ppm (d, 1H) 146.2 ppm 7.92 ppm (d, 1H) 141.3 ppm
Table 17: HSQC protons coupling to carbons A summary of the NMR results can be found in Table 18.
Literature search led to a linear furanocoumarin called swietenocoumarin B (Figure 16) first isolated by Bhide et al. (1977) from Chloroxylon swietenia (Rutaceae). However, no 13 C NMR data were published. Swietenocoumarin B has a methoxy group in position 5 and is prenylated in position 8. It has not been reported yet from T. montana.
40 Results and discussion
Position δ 13 C (ppm) δ 1H (ppm) J (Hz)
C2 160.6 s C3 114.0 d 6.32 d, 1H 10 C4 141.3 d 7.92 d, 1H 9.6 C4a 144.1 s C5 131.3 s C6 147.6 s C7 125.78* s C8 125.82* s C8a 114.3 s C1' 146.2 d 7.63 d, 1H 2.4 C2' 105.9 d 6.81 d, 1H 2.4 5 OCH 3 61.6 q 4.19 s, 3H C1'' 28.2 t 3.65 d, 2H 6.4 C2'' 122.2 d 5.08 m, 1H C3'' 133.0 s C4'' 25.7 q 1.66 s, 3H C5'' 18.3 q 1.81 s, 3H
* exchangeable
Table 18: NMR data of swietenocoumarin B in CDCl 3
Figure 17: 1H NMR spectrum of swietenocoumarin B
41 Results and discussion
Figure 18: 13 C NMR spectrum of swietenocoumarin B
Figure 19: COSY spectrum of swietenocoumarin B
42 Results and discussion
Figure 20: HSQC spectrum of swietenocoumarin B
Figure 21: HMBC spectrum of swietenocoumarin B
43 Results and discussion
The bioassay results (Table 19) from these two fractions showed again a considerable activity against the monocot bentgrass, while the dicot lettuce was almost not affected.
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V7 1 mg/mL DCM 7 2 3
AS1WS37V7B 1 mg/mL DCM 8 1 3 AS1WS37V7ppt 1 mg/mL DCM 7 2 3
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 19: Lettuce and bentgrass bioassay results of fraction V7, isolated compound V7B and fraction V7ppt
In the bioassay against Lemna (Figure 22) swietenocoumarin B showed no evaluable activities at lower concentrations than 1000 M. The evaluated data after 4 days yielded a curve where the IC 50 lied higher than 1000 M. After 7 days the data was scattered between 380% and 520%, therefore, the software was not able to evaluate an IC 50 .
44 Results and discussion
Figure 22: Dose-response curve of swietenocoumarin B
4.5 AS1WS37_V8_ppt (Bergapten)
OCH3
5 4 2' 6 3 4a 1' 8a 2 7 O O O 8 1 Figure 23: Bergapten
Molecular weight: 216.19
Molecular formula: C 12 H8O4
Fraction V8ppt turned out to be pure after the first silica gel column. 1H and
13 C NMR data were obtained in DMSO d 6 because of solubility problems with
45 Results and discussion
CDCl 3 and MeOD, respectively. LC MS indicated its molecular weight and the molecular formula. These results showed that fraction V8ppt was also a linear furanocoumarin with a methoxy group on one side. From literature search it turned out to be 5 methoxy psoralen called bergapten (Figure 23). The evaluated NMR data is in complete agreement with the published data (Table 24) (Khan et al., 2003).
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 160.2 160.3 C3 112.2 112.8 6.29 1H, d 6.34 1H, d C4 139.5 139.4 8.16 1H, d 8.17 1H, d C4a 105.7 106.7 C5 149.4 149.6 C6 112.3 113.0 C7 157.8 158.2 C8 93.1 94.0 7.31 1H, s 7.32 1H, s C8a 152.1 152.7 C1' 145.9 145.0 8.03 1H, d 8.02 1H, d C2' 105.5 105.3 7.39 1H, d 7.38 1H, d 5 OMe 60.2 61.1 4.25 3H, s 4.24 3H, s
Table 20: NMR-data (DMSO-d 6) of bergapten compared to Khan et al. (2003) As mentioned above, bergapten is a linear furanocoumarin with a methoxy group at position 5. It is used in external suntan preparations due to its ability to absorb near UV.
In the performed bioassay (Table 21) bergapten showed nearly complete growth inhibition of the bentgrass sample, while lettuce was almost not affected at all. In 2004, Hale et al. evaluated the phytotoxic properties of 5 methoxypsoralen in the same bioassays and showed inhibition of bentgrass at 30 M (Hale et al., 2004).
46 Results and discussion
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass AS1WS37V8ppt 1 mg/mL DCM 7 1 4
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 21: Lettuce and bentgrass bioassay results of bergapten
In the quantitative bioassay (Figure 24) bergapten showed negligable activity. Although Lemna growth was inhibited at lower concentrations than 1000 M, the software could not determine an IC 50 .
Figure 24: Dose-response curve of bergapten
47 Results and discussion
4.6 AS1WS37_V9_A_P2 (Psoralen)
5 4 2'' 6 3 4a 1 ' 8a 2 O 7 O O 8 1 Figure 25: Psoralen
Molecular weight: 186.16
Molecular formula: C 11 H6O3
Psoralen is a linear furanocoumarin with no substituents at all. Its pharmacological use lies in the treatment of vitiligo and psoriasis. The respective treatment is called PUVA (2.4.3 ).
Psoralen was identified from a literature database included in the GC MS software together with 1H and 13 C NMR in comparison with data in the literature (Table 22) (Razdan et al., 1987).
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 161.3 161.1 C3 114.8 114.7 6.34 1H, d 6.33 1H, d C4 144.3 144.2 7.77 1H, d 7.76 1H, d C4a 115.6 115.6 C5 120.0 120.0 7.43 7.46 1H, s C6 125.1 125.0 C7 156.6 156.2 C8 100.0 99.57.66 1 1H, s 7.29 1H, s C8a 152.2 152.2 C1' 147.1 147.07.66 1 1H, d 7.60 1H, d C2' 106.6 106.6 6.80 1H, d 6.80 1H, d
1 peaks overlapping
Table 22: NMR-data (CDCl 3) of psoralen compared to Razdan et al. (1987)
48 Results and discussion
Psoralen showed very high activity in the bioassay against bentgrass (Table 24) where growth or germination were completely inhibited. The dicot lettuce was affected more strongly than by any of the previously discussed compounds.
In the Lemna bioassay (Figure 26) psoralen yielded good results, as well. After both day 4 and day 7 considerable growth inhibition could be seen. The software determined an IC 50 at 115.11 M.
400 Day 4 Day 7
300
200
100 Lemna growth (increase in surface area) (increaseinsurface growth Lemna
0
0 0.1 1 10 100 1000
Concentration (uM) Figure 26: Dose-response curve of psoralen
49 Results and discussion
4.7 AS1WS37_V9_C (Dehydrogeijerin)
4'
3' 2' 5'
1' 5 4 6 3 O 4a
8a 2 7 H CO O O 3 8 1 Figure 27: Dehydrogeijerin
Molecular weight: 258.27
Molecular formula: C 15 H14 O4
While subfraction B was still a mixture of several compounds, subfraction C from fraction V9 turned out to be pure from its 1H and 13 C NMR, GC MS and LC MS data. The latter provided the molecular weight and the molecular formula. Complete identification was achieved by comparing the evaluated data with those from literature (Table 28) (Reisch & Bathe, 1988). A possible structure was suggested by the database included in the GC MS software.
50 Results and discussion
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 160.7 160.2** C3 114.0 113.7 6.25 d, 1H 6.30 d, 1H C4 143.5 143.2 7.63 d, 1H 7.63 d, 1H C4a 112.3 112.0 C5 130.5 130.2 7.68 s, 1H 7.78 s, 1H C6 128.4 128.1 C7 160.9 160.6** C8 99.8 99.5 6,79 s, 1H 6.85 s, 1H C8a 157.5 157.2* C1' 190.7 190.3 C2' 124.9 124.7 6.57 s, 1H 6.62 m, 1H C3' 156.9 156.3* Me cis 21.6 21.2 1.95 s, 3H 2.00 s, 3H Me trans 28.3 27.9 2.20 s, 3H 2.26 s, 3H OMe 56.4 56.1 3.90 s, 3H 3.97 s, 3H */** exchangeable
Table 23: NMR-data (CDCl 3) of dehydrogeijerin compared to Reisch & Bathe (1988)
Dehydrogeijerin (Figure 27) is a substituted simple coumarin, also called 6 senecionylherniarin. It has a methoxy group in position 7 and is alkylated with a senecionyl group in position 6 (Reisch & Bathe, 1988). It was first isolated from Geijera parviflora Lindl. (Rutaceae) (Lahey & MacLeod, 1967), and it was found in Thamnosma texana (Dominguez et al., 1984) but not in Thamnosma montana , being the second constituent isolated during this project new to this plant.
The bioassay results shown in Table 24 indicated a high acitivity against both bentgrass and lettuce. The activity against bentgrass was very strong, even at a concentration of 0.1 mg/mL. It was demostrated that the pure compounds showed stronger activity as the crude extract.
51 Results and discussion
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass
AS1WS37V9 1 mg/mL DCM 7 3 4
AS1WS37V9AP2 1 mg/mL DCM 7 3 5 AS1WS37V9C 1 mg/mL DCM 7 3 4 AS1WS37V9C 0.1 mg/mL DCM 7 1 2
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 24: Lettuce and bentgrass bioassay results of fraction V9 and of the isolated compounds
Dehydrogeijerin did not show any evaluable activity below 1000 M in the quantitative bioassay against Lemna (Figure 28). No IC 50 could be evaluated.
Day 4 500 Day 7
400
300
200
Lemna growth (increase in surface area) surface in (increase growth Lemna 100
0
0 0.1 1 10 100 1000
Concentration (uM) Figure 28: Dose-response curve of dehydrogeijerin
52 Results and discussion
4.8 AS1WS37_V10_C_cry (Isopimpinellin)
OCH3
5 4 2' 6 4a 3
1' 2 O 7 8a 8 O O 1
OCH3 Figure 29: Isopimpinellin
Molecular weight: 246.22
Molecular formula: C 13 H10 O5
V10 yielded 1.6 g of a mixture of ca. four compounds showing high activity in the prescreening.
V10C crystallized during the separation process and was recrystallized to yield V10Ccry. LC MS provided the molecular weight and the molecular formula. Literature research suggested the presence of another linear furanocoumarin with two methoxy groups, as seen in the 1H NMR spectra. Comparison of the recorded NMR spectra with published values confirmed the structure to be isopimpinellin (Figure 29), a 5,8 dimethoxypsoralen derivative (Razdan et al., 1987). A list of the compared values can be found in Table 25. Isopimpinellin has been isolated from T. montana before (Bennet & Bonner, 1953).
53 Results and discussion
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 160.6 160.7 C3 112.6 112.9 6.17 d, 1H 6.25 d, 1H C4 139.6 139.3 8.01 d, 1H 8.08 d, 1H C4a 107.4 107.1 C5 144.3 144.4 C6 114.6 114.9 C7 150.0 149.9 C8 127.9 128.3 C8a 143.6 143.7 C1' 145.1 146.0 7.55 d, 1H 7.59 d, 1H C2' 105.3 105.1 6.94 d, 1H 6.96 d, 1H OMe 61.7 61.5 4.10 s, 3H 4.14 s, 3H OMe 60.8 59.3 4.07 s, 3H 4.10 s, 3H
Table 25: NMR-data (CDCl 3) of isopimpinellin compared to Razdan et al. (1987)
The bioassay against lettuce and bentgrass revealed a high activity against both monocots and dicots (Table 28), similar to dehydrogeijerin. Bennet and Bonner analyzed the growth inhibitory activity of isopimpinellin against tomato seedlings in the 1950ies. A concentration of 9 mg/L caused general inhibition of the tomato seedling growth (Bennet & Bonner, 1953).
Isopimpinellin was the second tested compound that yielded evaluable data in the Lemna bioassay (Figure 30). After both day 4 and day 7 Lemna growth was influenced in a considerable way. The software determined an IC 50 at 82.47 M.
54 Results and discussion
Figure 30: Dose-response curve of isopimpinellin
4.9 AS1WS37_V10_E_B1 (Epoxysuberosin)
4'
3' 2' 5' O 5 4 1' 4a 3 6
7 2 8a H3CO O O 8 1 Figure 31: Epoxysuberosin
Molecular weight: 260.29
Molecular formula: C 15 H16 O4
55 Results and discussion
Subfraction V10E yielded two pure compounds. After the separation process, literature search revealed possible structures and comparing the recorded 1H NMR data with published values led to structure confirmation of epoxysuberosin (Dreyer et al., 1972). 13 C NMR data has not been reported yet and is presented here for the first time (Table 26).
Epoxysuberosin is a simple coumarin belonging to the herniarin group, alkylated in position 6. The prenyl group contains an epoxy group. It was first reported in 1981 as a constituent from Coleonema album (Rutaceae) (Gray, 1981), but it has not been isolated from T. montana before being the third constituent new to this species.
δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature
161.4 s 7.60 d, 1H (J=9.2) 7.59 d, J=9 160.7 s 7.27 s, 1H 7.27 s 154.9 s 6.75 s, 1H 6.79 s 143.6 d 6.20 d, 1H (J=9.2) 6.23 d, J=9 128.6 d 3.87 s, 3H 3.97 s methoxy CH CH O 124.2 s 2.97 2.69 m, 3H 3.17 2.67 m 2 113.1 d 1.36 s, 3H 1.41 C methyl 112.1 s 1.30 s, 3H 1.37 C methyl 98.8 d 63.3 d 59.0 s 56.0 q 29.2 t 24.9 q 19.0 q
Table 26: Comparison of published 1H-NMR data with recorded data plus recorded 13 C- data epoxysuberosin
Table 28 indicates that epoxysuberosin suppresses growth of both tested plants. Both tested monocots and dicots showed almost no growth or germination, thus making epoxysuberosin the most potent growth inhibitor isolated during this project.
56 Results and discussion
In the quantitative bioassay (Figure 32) epoxysuberosin did not show evaluable activity at lower concentrations than 1000 M. No IC 50 could be determined.
Figure 32: Dose-response curve of epoxysuberosin
57 Results and discussion
4.10 AS1WS37_V10_E_B2 (Alloimperatorin methyl ether epoxide)
Figure 33: Alloimperatorin methyl ether epoxide
Molecular weight: 300.31
Molecular formula: C 17 H16 O5
The second isolated molecule out of subfraction V10E showed the highest molecular weight. 1H NMR data indicated the existence of a methoxy group and showed the typical coupling pattern of protons in the aromatic region. The proton data showed furthermore two methyl groups. Comparison of already published values led to complete confirmation of the constituent as alloimperatorin methyl ether epoxide (Abou Elzahab et al., 1992). Kutney et al. isolated this substance from T. montana in 1972 (Kutney et al., 1972).
58 Results and discussion
δ 13 C (ppm) Position δ 13 C (ppm) δ 1H (ppm) δ 1H (ppm) literature literature C2 160.3 160.1 s C3 105.7 105.6 d 6.26 d, 1H 6.34 d, 1H C4 141.5 141.1 d 8.01 d, 1H 8.04 d, 1H C4a 114.9 114.8 s C5 122.2 121.9 s C6 114.9 114.8 s C7 147.2 147.8 s C8 126.2 126.1 s C8a 143.8 143.9 s C1' 146.3 146.2 d 7.61 d, 1H 7.64 d, 1H C2' 114.0 114.2 d 6.82 d, 1H 6.84 d, 1H OCH3 61.3 61.3 q 4.14 s, 3H 4.20 s, 3H C1'' 29.0 29.0 t 2.60 d, 1H 2.80 dd, 1H C2'' 64.1 64.0 d 2.93 m, 1H 2.99 dd, 1H C3'' 59.5 59.3 s 3.31 d, 1H 3.32 dd, 1H CH3 19.1 19.1 q 1.22 s, 3H 1.26 s, 3H CH3 24.6 24.5 q 1.41 s, 3H 1.44 s, 3H
Table 27: NMR-data (CDCl 3) of alloimperatorin methyl ether epoxide compared to Abou-Elzahab et al. (1992)
The lettuce and bentgrass bioassay (Table 28) showed moderate growth inhibition for both monocots and dicots. All three isolated substances of fraction V10 indicated a high activity in the performed bioassays. As expected, it revealed potent phytotoxic constiuents, each showing moderate to high growth inhibitory properties.
Ranking Sample ID Tested conc. Solvent used Day Lettuce Bentgrass AS1WS37V10 1 mg/mL DCM 7 3 4
AS1WS37V10Ccry 1 mg/mL DCM 7 3 4 AS1WS37V10EB1 1 mg/mL DCM 6 4 4 AS1WS37V10EB2 1 mg/mL DCM 6 3 3
Ranking based on scale 0 to 5 0 = no effect 5 = no growth or germination
Table 28: Lettuce and bentgrass bioassay results of fraction V10 and of the isolated compounds
59 Results and discussion
In the quantitative bioassay alloimperatorin methyl ether epoxide did not yield evaluable data. There is no activity at lower concentrations than 1000 M making it not possible for the software to determine an IC 50 .
Figure 34: Dose-response curve of alloimperatorin methyl ether epoxide
60 Summary 5 Summary
This thesis describes the bioassay guided fractionation of the dichloromethane extract of the aerial parts of Thamnosma montana Torr. & Frém., a Rutaceae native to the southwestern United States. Thamnosma montana was collected and the aerial parts were extracted with dichloromethane by Dr. Wolfgang Schühly in 2001. In phytotoxic pre screenings performed in Oxford, MS, in August 2008, T. montana showed considerable activity. The crude extract was fractionated using silica gel column chromatography with ethyl acetate and hexane as mobile phase. The collected fractions were subjected to a qualitative bioassay against the monocot Agrostis stolonifera L. (Poaceae) and the dicot Lactuca sativa L. (Asteraceae). In this bioassay, phytotoxic properties of the single fractions were evaluated. Only active fractions were further investigated and eight active pure compounds were isolated. As separation methods silica gel and reversed phase thin layer chromatography as well as preparative thin layer chromatography was used. Furthermore, silica gel flash column chromatography and both analytical and preparative high performance liquid chromatography were applied. NMR, GC MS EI and LC MS ESI were used to elucidate the structures of the isolated compounds. Nine constituents were isolated of which eight showed activity in the qualitative bioassays. All these constituents were derivatives of coumarin, mostly prenylated furanocoumarins. Since Thamnosma montana was investigated thoroughly in the middle of last century, six of the nine isolated compounds have already been found in this species. Three compounds, swietenocoumarin B, dehydrogeijerin and epoxysuberosin and have not been described in Thamnosma montana yet. All compounds were subjected to a quantitative bioassay against the monocot Lemna paucicostata L. (Lemnaceae). The goal of these bioassays was to evaluate the phytotoxic properties in a dose response manner. To quantify the
61 Summary
activity a software should evaluate an IC 50 . As a result, isopimpinellin and psoralen showed activity below concentrations of 1000 M, making it possible to evaluate an IC 50 . The other seven constituents were not potent enough to inhibit the growth of L. paucicostata below concentrations of 1000 M.
62 Appendix
6 Appendix
6.1 Bibliography
Abou Elzahab, Mohamed M., Adam, Waldemar, Saha Möller, Chantu R. (1992). Synthesis of Furanocoumarin Type Potential Intercalative Alkylating and Oxidizing Agents of DNA Through Dimethyldiosirane Epoxidation of Imperatorin and Its Derivatives. Liebigs Annalen der Chemie, 731 733
Bennet, Edward L., Bonner, James (1953). Isolation of Plant Growth Inhibitors from Thamnosma montana . American Journal of Botany, 40 , 29 33
Benson, Lyman, Darrow, Robert A. (1981). Trees and Shrubs of the Southwestern Deserts. The University of Arizona Press, Tucson
Bhide, K. S., Mujumdar, R. B., Rama Rao, A. V. (1977). Phenolics from the Bark of Chloroxylon swietenia DC. Indian Journal of Chemistry, 15B , 440 444
Brahmachari, Goutam (2009). Natural Products: Chemistry, Bio chemistry and Pharmacology. Alpha Science International Ltd., Oxford
Bruneton, Jean (1995). Pharmacognosy, Phytochemistry, Medicinal Plants. Technique & Documentation, Paris
Chang, P. T. O., Cordell, G. A., Aynilian, G. H., Fong, H. H. S., Farnsworth, N. R. (1975). Alkaloids and Coumarins of Thamnosma montana . Lloydia, 39 , 134 140
63 Appendix
Chase, Mark W., Morton, Cynthia M., Kallunki, Jacquelyn A. (1999). Phylogenetic Relationships of Rutaceae: A Cladistic Analysis of the Subfamilies Using Evidence from rbcL and atpB Sequence Variation. American Journal of Botany, 86 , 1191 1199
Dayan, Franck E., Cantrell, Charles L., Duke, Stephen O. (2009). Natural products in crop protection. Bioorg. Med. Chem., In Press, Corrected Proof, Available online 27 January 2009
Dayan, Franck E., Romagni, Joanne G., Duke, Stephen O. (2000). Investigating the Mode of Action of Natural Phytotoxins. Journal of Chemical Ecology, 26 , 2079 2094
Dewick, Paul M. (2002). Medicinal Natural Products A Biosynthetic Approach. John Wiley & Sons, LTD, Chichester
Dominguez, X. A., R. Franco, J. Verdes S., A. Zamudio, E.Y. Guevara Z. (1984). Coumarins from desert rue Thamnosma texana (Gray) Torr. Revista Latinoamericana de Quimica, 15 , 138 139
Dreyer, D. L. (1966). Constituents of Thamnosma montana Torr. and Frem. Tetrahedron, 22 , 2923 2927
Dreyer, David L., Pickering, Michael V., Cohan, P. (1972). Distribution of Limonoids in the Rutaceae. Phytochemistry, 11 , 705 713
El Shafae, Azza M., Ibrahim, M. A. (2003). Bioactive kaurane diterpenes and coumarins from Fortunella margarita . Pharmazie, 58 , 143 144
Evans, William Charles (1999). Trease & Evans' Pharmacognosy. Saunders, London
64 Appendix
Gray, Alexander I. (1981). New Coumarins from Coleonema album . Phytochemistry, 20 , 1711 1713
Hale, Amber L.; Meepagala, Kumudini M.; Oliva, Anna; Aliotta, Giovanni; Duke, Stephen O. (2004). Phytotoxins from the Leaves of Ruta graveolens . Journal of Agricultural and Food Chemistry, 52 , 3345 3349
Jaeger, Edmund C. (1941). Desert Wild Flowers. Stanford University Press, Stanford
Kearney, Thomas H., Peebles, Robert H., Howell, John Thomas, McClintock, Elizabeth (1960). Arizona flora. University of California Press, Berkeley
Khan, Ikhlas A., Ngunde Ngwendson, J., Bedir, E., Efange, S. M. N., Okunji, C. O., Iwu, M. M., Schuster, B. G. (2003). Constituents of Peucedanum zenkeri seeds and their antimicrobial effects. Pharmazie, 58 , 587 589
Kutney, J. P., Inaba, T., Dreyer, D. L. (1970). Further Studies on Constituents of Thamnosma montana Torr. and Frem. The Structure of Thamnosin, a Novel Dimeric Coumarin System. Tetrahedron, 26 , 3171 3184
Kutney, J. P., Verma, A. K., Young, R. N. (1972). Studies on the Constituents of Thamnosma montana Torr. and Frem. The Structure of Thamnosmin, a Novel Coumarin Epoxide. Tetrahedron, 28 , 5091 5104
Kutney, James P., Young, Robert N., Verma, Ashok K. (1969). Novel Epoxides from Thamnosma montana Torr. and Frem.. Tetrahedron Letters, 23 , 1845 1847
Lahey, F. N., MacLeod, J. K. (1967). The Coumarins of Geijera parviflora LINDL. Australian Journal of Chemistry, 20 , 1943 1955
65 Appendix
Mabberley, D. J. (1997). The Plant Book. Cambridge University Press, Cambridge
Masuda, Takahiro, Takasugi, Mitsuo, Anetai, Masaki (1998). Psoralen and other Linear Furanocoumarins as Phytoalexins in Glehnia littoralis . Phytochemistry, 47 , 13 16
Michel, Albrecht, Johnson, Robert D., Duke, Stephen O. and Scheffler, Brian E. (2004). Dose Response Relationships Between Herbicides with Different Modes of Action and Growth of Lemna paucicostata : An Improved Ecotoxicological Method. Environmental Toxicology and Chemistry, 23 , 1074 1079
Moerman, Daniel E. (1998). Native American Ethnobotany. Timber Press, Inc., Portland
Rahman, Atta ur, Choudhary, M. Igbal, Thomsen, William J. (2001). Bioassay Techniques for Drug Development. Harwood Academic Publishers, Newark
Razdan, T. K., Qadri, B., Harkar, S., Waight, E. S. (1987). Chromones and Coumarins from Skimmia laureola . Phytochemistry, 26 , 2063 2069
Reisch, Johannes, Bathe, Andreas (1988). Synthese der Cumarine 6 und 8 Naphthoherniarin, Dehydrogeijerin und Murraol. Liebigs Annalen der Chemie , 543 547
Robinson, James W. (1987). Undergraduate instrumental analysis. Marcel Dekker, INC., New York
Shreve, Forrest, Wiggins, Ira L. (1964). Vegetation and Flora of the Sonoran Desert. Stanford University Press, Stanford
66 Appendix
Silverstein, R.M., Bassler, G. Clayton, Morrill, Terence C. (1991). Spectrometric identification of organic compounds. John Wiley & Sons, INC., Hoboken
USDA, NRCS. 2009. The PLANTS Database (http://plants.usda.gov, 24 February 2009). National Plant Data Center, Baton Rouge, LA 70874 4490 USA
6.2 Index of Figures
Figure 1: Distribution of the Rutaceae family (retrieved May 28 th 2009 http://www.mobot.org/MOBOT/research/APweb/maps/Rutaceae.gif)...... 2 Figure 2: Distribution of Thamnosma in North America (Plants, 2009)...... 3 Figure 3: Thamnosma montana whole plant (© jrdnz, retrieved May 28 th 2009 http://www.flickr.com/photos/jordanz/2282292007/)...... 4 Figure 4: Thamnosma montana flower (© Aaron Schusteff, retrieved May 28 th 2009 http://calphotos.berkeley.edu/cgi/img_query? query_src=&enlarge=0000+0000+1008+1450)...... 4 Figure 5: 1flowering branch with several dark, bluish purple flowers, the leaves are on only the young lateral branch; 2branch with fruits; 3the curious bispheroidal fruit covered with minute glands (Benson & Darrow, 1981)...... 5 Figure 6: Thamnosma montana fruit (© Aaron Schusteff, retrieved May 28 th 2009 http://calphotos.berkeley.edu/cgi/img_query? query_src=&enlarge=0000+0000+1008+1449)...... 5 Figure 7: Thamnosma montana habitat (© Rolf Muertter, retrieved May 28 th 2009 http://www.flickr.com/photos/muertter/839566581/)...... 5 Figure 8: Distribution of T. montana (Plants, 2009)...... 6 Figure 9: Flow chart of a typical bioassay guided fractionation (Rahman/Choudhary/Thomsen, 2001)...... 12 Figure 10: Flow chart of bioassay guided fractionation...... 18 Figure 11: Conduction of a bioassay...... 20 Figure 12: Isoimperatorin...... 33
67 Appendix
Figure 13: Dose response curve of isoimperatorin...... 35 Figure 14: Suberosin...... 35 Figure 15: Dose response curve of suberosin...... 37 Figure 16: Swietenocoumarin B...... 38 Figure 17: 1H NMR spectrum of swietenocoumarin B...... 41 Figure 18: 13 C NMR spectrum of swietenocoumarin B...... 42 Figure 19: COSY spectrum of swietenocoumarin B...... 42 Figure 20: HSQC spectrum of swietenocoumarin B...... 43 Figure 21: HMBC spectrum of swietenocoumarin B...... 43 Figure 22: Dose response curve of swietenocoumarin B...... 45 Figure 23: Bergapten...... 45 Figure 24: Dose response curve of bergapten...... 47 Figure 25: Psoralen...... 48 Figure 26: Dose response curve of psoralen...... 49 Figure 27: Dehydrogeijerin...... 50 Figure 28: Dose response curve of dehydrogeijerin...... 52 Figure 29: Isopimpinellin...... 53 Figure 30: Dose response curve of isopimpinellin...... 55 Figure 31: Epoxysuberosin...... 55 Figure 32: Dose response curve of epoxysuberosin...... 57 Figure 33: Alloimperatorin methyl ether epoxide...... 58 Figure 34: Dose response curve of alloimperatorin methyl ether epoxide...... 60
6.3 Index of Tables
Bioassay results after first VLC...... 20 Solvent system for fraction V6...... 21 Bioassay results of V6 subfractions...... 22 Solvent system for fraction V7...... 23 Subfractions of V7...... 23 Bioassay results of V7 subfractions...... 24 Solvent system for fraction V9...... 25
68 Appendix
Subfractions of V9...... 25 Bioassay results V9 subfractions ...... 25 Subfractions of V10...... 26 Bioassay results V10 subfractions...... 27 Solvent system for subfraction V10E...... 28 HPLC method details V11...... 29 NMR data of isoimperatorin compared to Masuda et al. (1998)...... 34
NMR data (CDCl 3) of suberosin compared to El Shafae & Ibrahim (2003).....36 Lettuce and bentgrass bioassay results of fraction V6 and of the isolated compounds...... 37 HSQC protons coupling to carbons...... 40
NMR data of swietenocoumarin B in CDCl 3...... 41 Lettuce and bentgrass bioassay results of fraction V7, isolated compound V7B and fraction V7ppt...... 44
NMR data (DMSO d 6) of bergapten compared to Khan et al. (2003)...... 46 Lettuce and bentgrass bioassay results of bergapten...... 47
NMR data (CDCl 3) of psoralen compared to Razdan et al. (1987)...... 48
NMR data (CDCl 3) of dehydrogeijerin compared to Reisch & Bathe (1988)....51 Lettuce and bentgrass bioassay results of fraction V9 and of the isolated compounds...... 52
NMR data (CDCl 3) of isopimpinellin compared to Razdan et al. (1987)...... 54 Comparison of published 1H NMR data with recorded data plus recorded 13 C data epoxysuberosin...... 56
NMR data (CDCl 3) of alloimperatorin methyl ether epoxide compared to Abou Elzahab et al. (1992)...... 59 Lettuce and bentgrass bioassay results of fraction V10 and of the isolated compounds...... 59
69