Phytochemical investigation of the roots of Peltodon longipes and in vitro cytotoxic studies of abietane diterpenes
INAUGURALDISSERTATION
zur Erlangung des Doktorgrades der Fakultät für Chemie, Pharmazie und Geowissenschaften der Albert-Ludwigs-Universität Freiburg im Breisgau
vorgelegt von
Marcio Fronza aus Tucunduva, RS (Brasilien)
June 2011
Dekan: Prof. Dr. Harald Hillebrecht
Leiterin der Arbeit: Prof. Dr. Irmgard Merfort
Referentin: Prof. Dr. Irmgard Merfort
Korreferent: Prof. Dr. Stefan Laufer
Drittprüfer: Prof. Dr. Andreas Bechthold
Tag der Verkündigung des Prüfungsergebnisses: 15-07-2011
Parts of this work have been published in the following conference presentations and publications:
Publications
Fronza M., Lamy E., Günther S., Murillo R., Heinzmann B., Laufer S., Merfort I. In vitro cytotoxic molecular mechanism studies of abietane diterpenes from Peltodon longipes . In preparation.
Fronza M., Murillo R., Ślusarczyk S., Adams M., Hamburger M., Heinzmann B., Laufer S., Merfort I. In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and S. sahendica . Bioorganic & Medicinal Chemistry, in press .
Geller F., Schmidt C., Gottert M., Fronza M., Heinzmann B., Flores E.M., Merfort I., Laufer S. Identification of rosmarinic acid as the major active compound constituent in Cordia americana . Journal of Ethnopharmacology . v.130, p.333 - 338, 2010.
Schmidt C. A., Fronza M., Gottert M., Geller F., Laufer S., Merfort I. Biological studies on Brazilian plants used in wound healing. Journal of Ethnopharmacology . v.122, p.523 - 532, 2009.
Fronza M., Heinzmann B., Hamburger M., Laufer S., Merfort I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts. Journal of Ethnopharmacology . v.126, p.463 - 467, 2009.
Conferences - poster presentation
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort I. Antiproliferative activity of diterpenes from Peltodon longipes . In : Deutsch-Brasilianisches Jahr 2010/11- Workshop: Drugs from Natural Sources - São Paulo, SP, Brazil, 2010.
Schmidt C.A., Fronza M., Murillo R., Brecht V., Heinzmann B., Laufer S.A., Merfort, I. Wound Healing Properties of Catechin Derivatives Isolated from Barks of Parapiptadenia rigida . In : Deutsch-Brasilianisches Jahr 2010/11- Workshop: Drugs from Natural Sources - São Paulo, SP, Brazil, 2010.
Geller F., Schmidt C., Goettert M., Fronza M., Heinzmann B., Werz O., Merfort I., Laufer S. Rosmarinic acid as the effective compound in Cordia americana . In : Deutsch-Brasilianisches Jahr 2010/11- Workshop: Drugs from Natural Sources - São Paulo, SP, Brazil, 2010.
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort I. Antiproliferative activity of diterpenes from Peltodon longipes . In : XIX° Congreso Italo-Latinoamericano de Etnomedicina, Cagliari (Villasimius), Italy, 2010.
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort, I. Abietane diterpenoids from the roots of the Lamiaceae Peltodon longipes preferentially inhibit topoisomerase-I. In : 22 nd Irsser Naturstofftage - Aktuelle Entwicklungen in der Naturstoff-Forschung, Irsee - Germany, 2010.
Gottert M., Fronza M., Schmidt C. A., Geller F., Heinzmann B., Merfort I., Laufer S. Effect of natural phenolic compounds on p38 α MAPK activity. In : IV Simpósio Brasil-Alemanha / 4. Deutsch-Brasilianisches Symposium, Curitiba-Brazil, 2009.
Fronza M., Gottert M., Schmidt C. A., Heinzmann B., Jaroszewski J.W., Laufer S., Merfort I. Isolation of cytotoxic diterpenes from the lipophilic extract of Peltodon longipes. In : IV Simpósio Brasil-Alemanha / 4. Deutsch-Brasilianisches Symposium, Curitiba-Brazil, 2009.
Schmidt C. A., Fronza M., Gottert M., Murillo R., Heinzmann B., Laufer S., Merfort I. New catechin derivates from Parapiptadenia rigida. In : IV Simpósio Brasil-Alemanha / 4. Deutsch-Brasilianisches Symposium, Curitiba-Brazil, 2009.
Fronza M., Gottert M., Murillo R., Laufer S., Merfort I. Abietane diterpenoids from the roots of Peltodon longipes with cytotoxic activity and inhibitory activity towards p38 α MAPK. In: 6 th Status Seminar Chemical Biology - DECHEMA - Natural products and nucleic acids as chemical tools, Frankfurt - Germany, 2009.
Geller F., Gottert M., Fronza M., Schmidt C.A., Heinzmann B., Merfort I., Laufer S. Phytochemical and biological investigation on the ethanolic extract of Cordia americana. In : 6 th Status Seminar Chemical Biology - DECHEMA - Natural products and nucleic acids as chemical tools, Frankfurt-Germany, 2009.
Fronza M., Gottert M., Schmidt C. A., Heinzmann B., Jaroszewski J.W., Laufer S., Merfort I. Diterpenes from Peltodon longipes with caspase-3 activity. In: Drug Discovery and Delivery: Membrane Proteins and Natural Product Research, Freiburg - Germany, 2009.
Gottert M., Fronza M., Schmidt C. A., Geller F., Merfort I., Laufer S. Natural phenolic compounds as inhibitors of P38 α MAPK. In : Drug Discovery and Delivery: Membrane Proteins and Natural Product Research, Freiburg-Germany, 2009.
Schmidt C.A., Fronza M., Gottert M., Murillo R., Heinzmann B., Laufer S., Merfort I. Two new catechin derivatives from Parapiptadenia rigida . In : Drug Discovery and Delivery: Membrane Proteins and Natural Product Research, Freiburg-Germany, 2009.
Schmidt C.A., Fronza M., Gottert M., Murillo R., Heinzmann B., Laufer S., Merfort I. Phytochemical and biological studies of the bark from Parapiptadenia rigida. In: 2 nd BCNP - Brazilian Conference on Natural Products, São Pedro - Brazil, 2009.
Gottert M., Luik S., Fronza M., Geller F., Schmidt C.A., Merfort I., Laufer S. Biological testing of bioactive compounds that inhibit p38 MAPK. In : 5 th Status Seminar Chemical Biology - DECHEMA - Chemical Biology in Europe: Research across disciplines and countries, Frankfurt-Germany, 2008.
Fronza M., Geller F., Bittencourt C., Flores E.M.M., Heinzmann B., Laufer S., Merfort I. The scratch assay: a suitable in vitro tool for studying wound healing effects. In : 7 th Joint Meeting of the Association Francophone pour l'Enseignement et la Recherche en Pharmacognosie (AFERP), American Society of Pharmacognosy (ASP), Society for Medicinal Plant Research (GA), Phytochemical Society of Europe (PSE), and Societ Italiana di Fitochimica (SIF), Athens - Greece, 2008.
Conferences - short talks
Fronza M., Merfort I. Antiproliferative activity of diterpenes from Peltodon longipes . In : XIX° Congreso Italo-Latinoamericano de Etnomedicina, Cagliari (Villasimius), Italy, 2010.
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort I. Cytotoxic abietane diterpenes from Peltodon longipes and their mode of action. In : 58 th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research and 7th Tannin Conference, Berlin - Germany, 2010.
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort I. Abietane diterpenoids from the roots of the Lamiaceae Peltodon longipes preferentially inhibit topoisomerase-I. In : 22 nd Irsser Naturstofftage - Aktuelle Entwicklungen in der Naturstoff-Forschung, Irsee - Germany, 2010.
Fronza M., Günther S., Murillo R., Lamy E, Heinzmann B., Laufer S., Merfort I. Diterpenoids from a Brazilian medicinal plant preferentially inhibit topoisomerase-I. In : 7 th Immunology meeting, Freiburg - Germany, 2010.
Fronza M., Heinzmann B., Geller F., Laufer S., Merfort I. An improved scratch assay for studying the wound healing effects of medicinal plants. In : IV Simpósio Brasil-Alemanha / 4. Deutsch-Brasilianisches Symposium, Curitiba-Brazil, 2009.
Index I
1. Introduction...... 1
1.1. Natural products as leads for the development of potential drugs...... 1
1.2. Peltodon longipes A.St.-Hil. ex Benth...... 6
1.3. Diterpenes ...... 8
1.3.1. Abietane diterpenes...... 11
1.4. Aim of the thesis ...... 24
2. Results ...... 25
2.1. Scratch assay ...... 25
2.2. Studies of Brazilian medicinal plants on HNE...... 32
2.3. Peltodon longipes ...... 34
2.3.1. Isolation procedure...... 34
2.3.2. Structure elucidation of the isolated abietane diterpenes...... 35
2.3.2.1 7 α-acetoxyroyleanone (syn. 7-O-acetylhorminone)(1)...... 35
2.3.2.2 7 α-hydroxyroyleanone (syn horminone) (2)...... 45
2.3.2.3 Royleanone (3)...... 53
2.3.2.4 7-ketoroyleanone (syn 7-oxoroyleanon) (4)...... 61
2.3.2.5 7 α-ethoxyroyleanone (5)...... 68
2.3.2.6 Iguestol (6) ...... 76
2.3.2.7 Deoxyneocryptotanshinone (7) ...... 84
2.3.2.8 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8)...... 92 Index II
2.3.2.9 Inuroyleanol (9)...... 100
2.3.2.10 Sugiol (10)...... 108
2.3.2.11 Cryptojaponol (11) ...... 116
2.3.2.12 Orthosiphonol (12)...... 124
2.3.3. HPLC analysis...... 132
2.3.4. Biological studies...... 133
2.3.4.1 Cytotoxic studies of diterpenes from P. longipes and from Salvia species using the MTT assay...... 133
2.3.4.2 Studies on the alkylating properties of compounds 1 - 4 and 10 ...... 141
2.3.4.3 Studies on human DNA topoisomerase I and II inhibitory activity...... 143
2.3.4.4 Molecular modeling studies ...... 146
2.3.4.5 Cell cycle analysis...... 148
2.3.4.6 Apoptotic activity using caspase-3 like assay...... 151
2.3.4.7 Cell death detection ELISA...... 153
2.3.4.8 Apoptotic cell death measured by flow cytometry...... 154
2.3.4.9 Protein kinase inhibitory activity ...... 155
2.3.4.10 Determination of DNA damage and repair using the alkaline comet assay……...... 156
2.3.4.11 MTT assay with pan-caspase inhibitor (Q-VD-OPH)...... 159
2.3.4.12 MTT studies with the ROS-scavenging BHA...... 161
2.3.4.13 ROS quantification analysis...... 163 Index III
2.3.4.14 Inhibitory activity on p38 α MAP Kinase...... 163
3. Discussion...... 165
3.1. Wound healing effects of traditional medicinal plants using the scratch assay 165
3.2. Peltodon longipes ...... 168
3.3. Biological studies of abietane diterpenes ...... 169
3.3.1. Cytotoxic studies of abietanes from Peltodon and Salvia Species...... 169
3.3.2. Studies on the alkylating properties of diterpenes from Peltodon longipes .. 170
3.3.3. Diterpenes from Peltodon longipes and their effects on topoisomerases ..... 171
3.3.4. Diterpenes from Peltodon and their effects on cell cycle ...... 178
3.3.5. Diterpenes possess further biological activities ...... 179
4. Summary...... 182
5. Materials and Methods...... 185
5.1. Materials ...... 185
5.1.1. Chemicals...... 185
5.1.2. Cytokines, antibodies, enzymes and kits...... 187
5.1.3. Consumable...... 188
5.1.4. Equipment ...... 190
5.1.5. Plant material...... 192
5.1.6. Computer Software’s and Databases...... 193
5.2. Methods...... 194 Index IV
5.2.1. Chromatographic and spectroscopic methods...... 194
5.2.2. Plant Extraction and Isolation Methods...... 197
5.2.3. Biological Methods ...... 204
5.2.3.1 Cell culture...... 204
5.2.3.2 Human Neutrophil Elastase assay...... 206
5.2.3.3 Scratch assay ...... 207
5.2.3.4 Cytotoxic studies...... 208
5.2.3.5 Caspase-3 like assay...... 210
5.2.3.6 Cell death detection ELISA...... 211
5.2.3.7 Quantification of ROS by dichlorofluorescin fluorescence assay ...... 212
5.2.3.8 Alkylating activity...... 212
5.2.3.9 Topoisomerases relaxation assays...... 213
5.2.3.10 Alkaline comet assay...... 215
5.2.3.11 FACS and Cell cycle distribution analysis...... 216
5.2.3.12 Protein kinase assay ...... 217
5.2.3.13 p38 α MAPK assay ...... 219
5.2.4. Docking studies...... 220
5.2.5. Statistical analysis ...... 221
6. References ...... 222
7. List of figures...... 237 Index V
8. List of tables...... 243
9. List of abbreviations ...... 245
10. Acknowledgment...... 248
11. Curriculum vitae ...... 250
Introduction 1
1. Introduction
1.1. Natural products as leads for the development of potential drugs
Natural products, especially from plants, have formed the basis of sophisticated traditional medicine systems that have been explored since thousands of years and an impressive number of drugs have been developed from them. The oldest information about plants used as drugs are from Mesopotamia and date from about 2600 BCE, while Egyptian medicines date from 1550 BCE with the best pharmaceutical records being the famous Ebers Papyrus which documented over 700 drugs. The Chinese Materia Medica has been extensively documented over the centuries, with the first record containing 52 prescriptions, dating from about 1100 BCE. Likewise, documentation of the Indian Ayurvedic system dates from approximately 1000 BCE (Potterat & Hamburger 2006; Samuelsson G & Bohlin L 2010; Dev 1999; Butler MS 2008). The Greeks and Romans also made substantial contributions to the rational development of the use of herbal drugs, with Dioscorides, a Greek physician (100 CE), accurately recording the collection, storage, and use of medicinal herbs during his travels with Roman armies throughout the “known world”, and Galen (130-200 CE), a practitioner and teacher of pharmacy and medicine in Rome, being well-known for his complex prescriptions and formulas used in compounding drugs. The preservation of the Greco-Roman expertise during the Dark and Middle Ages (5 th - 12 th centuries) may be attributed to the Arabs, who expanded it and included the use of their own resources together with Chinese and Indian herbs unknown to the Greco-Roman world (Cragg et al. 2009; Potterat & Hamburger 2006). The role played by plant-based systems in the healthcare of many different cultures has been continuing extensively investigated. The World Health Organization (WHO) has estimated that approximately 65% of the world’s population relies mainly on plant-derived traditional medicines for their primary health care (Fabricant & Farnsworth 2001; Butler MS 2008). Natural products are still being used direct or indirect as sources of leads to drugs against all classes of diseases. The goals of using plants as sources of therapeutics agents are 1) to use the whole plant or a part of it as a herbal remedy ( e.g. cranberry, echinacea, garlic, ginkgo 2 Introduction biloba, St. John’s wort, Arnica); 2) to isolate bioactive compounds for direct use as drugs (digoxin, morphine, reserpine, taxol, vinblastine, vincristine); 3) to isolate bioactive compounds of novel or known structures to be used as lead compounds that could be modified by semisynthesis to compounds with higher activity and/or lower toxicity (metformin, nabilone, taxotere, teniposide, verapamil); and 4) to use agents as pharmacologic tools (lysergic acid diethylamide, mescaline, yohimbine) (Fabricant & Farnsworth 2001; Farnsworth et al. 1985). Natural compounds offer huge structural diversity and in some cases great biological activity. Often chemical synthesis is not possible or too time consuming. The biodiversity on our planet is impressive. Plants comprise groups with about 250,000 species, however, only about 15% have been investigated phytochemically and 6% have been studied for biological activity (Verpoorte 2000). With high throughput screening methods becoming more advanced and available, the number of investigated plants will increase (Potterat & Hamburger 2008). There are some broad starting points to select and obtain plant material of potential therapeutic interest. When screening for new bioactive compounds we should consider the past, present, and future value of employing information from plants used in traditional medical practices (ethnomedicine). Plants that have been used in traditional medicine are more likely to yield pharmacologically active compounds. In the field of anticancer activity, a correlation between biological activity and plants used in folklore has been demonstrated (Potterat & Hamburger 2008; Rates 2001; Gordaliza 2007). The traditional processes to obtain a pharmacologically active pure constituent from a plant extract has always been a long and tedious process requiring substantial amount of material, the expertise of technicians and financial resources. In general it comprehends several and consecutive steps of preparative chromatographic separation. Mostly, the process is expensive and time consuming. The separation performance is poor, at least in the initial fractionation steps which are typically performed by open column chromatography, however, this procedure has led to successful isolation of many bioactive molecules in the past. The loss of bioactivity in the course of purification processes is not uncommon and replication of fractionation is most of the times not practicable (Potterat & Hamburger 2008). Over the last decade, new technologies and more effective strategies have been developed and adequate for a high throughput environment. The impact of HPLC-coupled spectroscopy has been enormous. The concerted use of HPLC-DAD, - MS and - NMR has opened entirely new possibilities for the characterization of secondary metabolites in biological extracts. Introduction 3
These combined techniques can provide structural information or even absolute configuration of a molecule in few minutes, with relative low cost and time compared to the traditional methods (Clarkson et al. 2005; Newman & Cragg 2007). Developments in the field of NMR, in particular the advent of new probe technology at high magnetic fields, as well as the miniaturization processes in crystallography facilitated structural elucidation and amounts of less than a milligram becoming a rather routine process (Jaroszewski 2005b; Jaroszewski 2005a). While analysis, purification and structure elucidation of natural products have experienced a technological breakthrough over the last decade, tracking bioactivity in complex matrices such as plant extracts remains a high challenging task. Extracts are complex mixtures and one of the greatest challenges is the judicious interfacing of chemistry and biology in order to correlate chemical analysis with biochemical data. The development of various innovative methodologies with high sensitivity for the analysis of macromolecule-ligand interactions, and the online integration of immunochemical and enzymatic methods with chemo-analytical systems have been recently implemented and provided the technological basis for this purpose. New offline strategies such as HPLC-based activity profiling, directly applicable to a broad range of mechanism-based and cellular assays, have gained increasing popularity in the context of industrial natural product screening (Potterat & Hamburger 2006). Plants have a long history of use in the treatment of cancer. Many of the claims for efficacy in the treatment of cancer should be viewed with some skepticism because cancer, as a specific disease entity, is likely to be poorly defined in terms of folklore and traditional medicine. This is in contrast to other plant-based therapies used in traditional medicine for the treatment of afflictions such as malaria and pain, which are more easily defined, and where the diseases are often prevalent in the regions where traditional medicine systems are extensively used (Lee 2010; Cragg et al. 2009). Cancer is a major public health burden in both developed and developing countries. It was estimated to occur in the United States 1,529,560 new cases and 569,490 patients died in 2010. Deaths from pancreatic ductal adenocarcinoma, also known as pancreatic cancer, rank fourth among cancer-related deaths in the US. In 2010, the estimated incidence of pancreatic cancer in the US was 43,140 cases, and an estimated 36,800 patients died from the disease (Jemal et al. 2010). A comprehensive genetic analysis of 24 pancreatic cancers suggested that the mature pancreatic cancer cell carries on average of 63 genetic alterations per cancer. These alterations can be grouped in 12 core signaling pathways. These results, if confirmed in larger 4 Introduction studies, would indicate that pancreatic cancer is genetically very complex and heterogeneous. Pancreatic cancer shows a rapid growth and metastatic distribution and often chemoresistance is developed during drug therapy. Several genetic alterations have been identified in these lethal cancers, including those in the CDKN2A , SMAD4 , and TP53 tumor suppressor genes and in the KRAS oncogene. The discoveries of these genes have provided important insights into the natural history of the disease and have stimulated efforts to develop new diagnostics and encouraged the development of new effective therapeutic drugs (Jones et al. 2008; Mimeault et al. 2005; Aho et al. 2007). Efforts to obtain anticancer agents from plants started more than five decades ago and have been particularly successful. Over 60% of all cancer drugs are of natural origin and a majority of these compounds were obtained from higher plants. In this instance, natural origin is defined as natural products, derivatives of natural products or synthetic pharmaceuticals based on natural product models. Up to the early 1990´s, screening for new antitumor compounds was mainly based on cytotoxic testing against a broad panel of cancer cell lines grown in vitro and subsequent testing in vivo . With the identification of an increasing number of targets associated with particular cancers, emphasis has meanwhile shifted towards more specific assays involving enzymes and receptors required in the cell division and tumor growth. Cyclin dependent kinases, tyrosine kinase receptors and topoisomerase are examples of such new molecular targets which have been subjected to high throughput screening programs (Newman & Cragg 2007; Newman 2008). In the last decade, a number of important new commercialized drugs have been obtained from natural sources by structural modification of natural compounds, or by the synthesis of new compounds designed following a natural compound as a model. The search for improved cytotoxic agents continues to be an important line in the discovery of modern anticancer drugs. The huge structural diversity of natural compounds and their bioactivity potential have resulted in the isolation of several products from plants, marine flora and microorganisms that can serve as “lead” compounds for the improvement of their therapeutic potential by molecular modification. Additionally, semisynthesis processes of new compounds, obtained by molecular modification of the functional groups of lead compounds, are able to generate structural analogues with greater pharmacological activity and with fewer side effects. These processes, complemented with high-throughput screening protocols, combinatorial chemistry, computational chemistry and bioinformatics are able to afford compounds that are far more efficient than those currently used in clinical practice. Combinatorial biosynthesis is also Introduction 5 applied for the modification of natural microbial products. Likewise, advances in genomics and the advent of biotechnology have improved both the discovery and production of new natural compounds (Butler MS 2008; Cragg et al. 2009; Lee 2010). One example of these leads is podophyllotoxin, a natural cyclolignan with antiviral and antitumor properties. Podophyllotoxin is the most abundant lignan isolated from Podophyllin peltatum (Berberidaceae). Since ancient times, podophyllotoxin has been used for medicinal purposes as a cathartic and antihelminthic agent. The chemistry explored around this compound has led to the introduction in clinical practice of etoposide (and its pro-drug etopophos) and teniposide, drugs that are efficacious against solid tumor of the lung and colon and against leukaemia among other antineoplastic properties (Butler MS 2008; Itokawa et al. 2008; Hartmann & Lipp 2006). Other examples of natural lead anticancer compounds are molecules such as camptothecin, paclitaxel or vinblastine. These compounds and their derivatives showed extraordinary antineoplastic properties, some of them have being found among the most widely prescribed chemotherapeutic agents. The alkaloid camptothecin (CPT) was first isolated in 1966 from the wood of a tree found in Tibet and China named Camptotheca acuminata (Nyssaceae) in the course of the anticancer screening program under the auspices of the National Cancer Institute (NCI). Meanwhile, it has been obtained also from several other plants belonging notably to the families of Apocynaceae and Icacinaceae. Animal studies revealed a potent antitumor activity, however, poor water solubility and severe side effects resulted in the discontinuation of Phase II trials in 1972. The discovery of its mode of action in 1980s revived the interest in CPT. CPT inhibits topoisomerase I by binding to the topoisomerase I-DNA binary complex, thereby inducing single strand breaks of cellular DNA. Intensified efforts in medicinal chemistry have been made to modify CPT and led to the development of more effective analogues with increased efficacy, solubility and reduced side effects. Two of its derivatives have been commercialized namely, topotecan and irinotecan (Lee 2010; Potterat & Hamburger 2008; Butler MS 2008; Hartmann & Lipp 2006). Other natural anticancer products also have been subjected to structural modifications in the search of better anticancer drugs, such as mitomycin C, doxorubicin, daunorubicin, bleomycin, mitramicin, masoprocol and resveratrol among many others (Lee 2010). In summary, natural products play an important role in the development of drugs, especially for the treatment of cancer. Podophyllotoxin, vinblastine, camptothecin and paclitaxel are only few examples of natural anticancer drugs within the broad arsenal of 6 Introduction natural compounds whose structural modification has led to more potent and less toxic compounds than the prototype.
1.2. Peltodon longipes A.St.-Hil. ex Benth
Peltodon longipes A. St. - Hil. ex Benth belongs to the Lamiaceae family also known as the mint family. The original family name was Labiatae, due to the presence of flowers with petals fused into an upper lip and a lower lip. The Lamiaceae family is distributed worldwide and contains about 236 genera and 6,900 to 7,200 species. There are approximately 8 species in the genus Peltodon ; P. comaroides, P. longipes, P. pusillus, P. radicans, P. repens, P. rugosus, P. tomentosa and P. tomentosus (Harley et al. 2004). Little is known about the botany of Peltodon longipes . It is described as an herb with very often woody rootstock, vegetative shoots more or less procumbent, leaves mid green, strongly rugose and scabrid, flower in heads on long leafless scapes, bracts green tinged purple, calyces blackish purple and corola deep purple red. It is usually found in grassland on moist deep loan (Figure 1.1 and Figure 1.2) (Mentz et al. 1997).
Figure 1.1 - Peltodon longipes subshrub or herb. Introduction 7
Figure 1.2 - Flowers and roots of Peltodon longipes .
As observed in Figure 1.3, P. longipes has been found only in South America, mainly in Brazil, Argentina, Uruguay and Paraguay.
Adapted from : http://www.discoverlife.org/mp/20m?act=make_map
Figure 1.3 - Worldwide distribution of Peltodon longipes .
Peltodon longipes is locally known as “baicuru amarelo” or “hortela do mato” in south of Brazil and has been externally used in folk medicine mainly as an anti-inflammatory and antiseptic remedy. No phytochemical studies are described in the literature. Therefore, the compounds responsible for the pharmacological effects are still unknown (Mentz et al. 1997).
8 Introduction
1.3. Diterpenes
Diterpenes are a class of secondary metabolites with a large variety of structures. The skeleton of every diterpene in general contains 20 carbon atoms. However, additional groups can be linked to the diterpene skeleton by an oxygen atom increasing the carbon atom count to more than 20 per diterpene. Diterpenes represent the second largest class of terpenoids, with over 5000 compounds belonging to 200 distinct skeletal types (Dev 1989). Terpenoids, terpenes or isoprenoids are a widespread class of natural products derived from a common biosynthetic pathway based on the mevalonate as parent. The diverse structural types were rationalized by the “Biogenic Isoprene rule” of Ruzicka in 1953, which states that the carbon skeleton of the terpenes is formed from a linear arrangement of isoprene units (C5) followed by various cyclisations and re-arrangements of the carbon skeleton. The main classes of terpenes are C10-monoterpenoids, C15-sesquiterpenoids, C20-diterpenoids, C25-sesterterpenoids, C30-triterpenoids, C40-carotenoids and C10 3-10 4 rubbers. In the diterpene group, the carbon skeletons are derived from a regular isoprene tetramer consisting of 4 isoprene units connected head to tail (Ruzicka 1953; Chinou 2005). The biosynthetic origin of this five-carbon unit (isoprene) was established via a common pathway, named the acetate-mevalonate pathway. As shown in Figure 1.4 the sequence starts with condensation of three molecules acetyl-CoA forming the important intermediate 3- hydroxy-3-methyl-glutaryl-CoA. The enzyme HMGCoA-reductase catalyses the formation of mevalonic acid (MVA) and is known to be a key enzyme of the acetate-mevalonate pathway. Isopentenyl diphosphate (IPP) is biosynthesized by subsequent phosphorylations and decarboxylation. IPP and its isomer dimethylallyl diphosphate activate biogenetic precursors of the linear branching point molecules in isoprenoid biosynthesis, geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate, respectively. However, the concept of a unique biosynthesis of isoprenic units is not generally accepted and other pathways for the formation of the isoprene skeleton should be considered (Dong et al. 2011; Ruzicka 1953; Chinou 2005). Introduction 9
O
SCoA 3x acetyl-CoA
OH
HOOC COSCoA
3-hydroxy-3- methylglutaryll-CoA
HMGCoA-reductase
OH
HOOC
OH R-Mevalonic acid
OPP OPP Isopentenyl Dimethylallyl diphosphatase (IPP) diphosphatase (DMAPP)
+ IPP
Monoterpenes Geranyl diphosphatase
+ IPP
Monoterpenes Farnesyl diphosphatase Triterpenes + IPP
Diterpenes Geranylgeranyl diphosphatase Tetraterpenes
Figure 1.4 - Isoprenoid biosynthesis via the acetate-mevalonate pathway (adapted from Chinou 2005).
The diterpenes (C-20) constitute a vast class of isoprenoid natural products, biosynthesized from mevalonic acid through geranylgeranyl diphosphate. They are classified according to the number of ring systems present. In addition, they form either acyclic or macrocyclic substances. Many diterpenes have additional ring systems, which occur in side chains, in ester substitutions or as epoxides. Some have 6-membered rings that have undergone aromatization, while others have fused 5- and 7- member rings. As observed in Figure 1.5, they can be divided in acyclic (phytanes), bicyclic (labdanes, clerodanes), tricyclic (pimaranes, abietanes), tetracyclic (kauranes, gibberellanes), macrocyclic diterpenes (taxanes, cembranes, tiglianes, lathyranes) and mixed compounds, in accordance with the number and the cyclization patterns displayed by their skeletons. They are found mainly in plants and fungi, although diterpenes have also been detected in marine organisms and insects as well (Garcia et al. 2007; Hanson 2007; Hanson 2009; Hoffmann 2006). 10 Introduction
Acyclic Diterpene
Phytane
Bicyclic Diterpenes
Labdane Clerodane
Tricyclic Diterpenes
Primarane Abietane
Tetracyclic Diterpenes
Kaurane Gibberellane
Macrocyclic Diterpenes
Cembrane Lathyrane
Taxane Tigliane
Figure 1.5 - Classification of diterpenes according to the number of ring systems present (Hoffmann 2006).
Introduction 11
1.3.1. Abietane diterpenes
Chemically, the abietane-type diterpenes are generally composed of three rings, including naphthalene rings A and B. Among this class of diterpenes tanshinones deserve special interest. They possess an ortho - or para - quinone or lactone ring C, and a furan or dihydrofuran ring D. They are widely distributed and mainly found in the Lamiaceae family and have been repeatedly isolated from different Salvia species (Dong et al. 2011; Garcia et al. 2007). Abietane diterpenes have attracted particular attention from the scientific community because they have shown a broad spectrum of pharmacological activity such as antibacterial, antileishmanial, antifungal, anti-inflammatory, trypanocidal, antioxidant, cardiovascular and cytotoxic, among many other.
Antibacterial activity Since ancient times several herbal extracts have been reported to possess antimicrobial activity. The antimicrobial activity of many abietane diterpenes have been tested against standard bacterial strains such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Bacillus subtilis, Escherichia coli , Proteus mirabilis and Pseudomonas aeruginosa. In general, abietane diterpenes have shown antibacterial activity mainly against S. aureus .
The widely found horminone and 7α-acetoxyroyleanone have been demonstrated to be active against Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis . Horminone showed an minimum inhibitory concentration (MIC) against S. epidermidis in a range of 1.5 to 31.25 µg/ml; against B. subtilis from 1.5 to 62.5 µg/ml, and against S. aureus from 6.5 to 62.5 µg/ml. Horminone was also active against Enterococcus faecalis with a MIC of 14 µg/ml. 7 α-acetoxyroyleanone exhibited similar results being more active against B. subtilis with a MIC of 3 µg/ml (Ulubelen et al. 2001; Batista et al. 1994; Ulubelen et al. 1999a; Topcu & Goren 2007). Antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) of several oxidized natural abietane diterpenes were studied by Yang et al. 2001. Demethylcryptojaponol, salvinolone, 12-hydroxyabieta- 8,11,13-trien-6-one, 6 β-hydroxyferruginol and taxodione showed potent activity against both bacteria strains with a MIC from 4 to 10 g/ml for MRSA and with a MIC between 4 to 16 12 Introduction
g/ml for VRE, respectively. Quinone methide, 11-hydroxy-12-oxo-7,9(11),13-abietatriene, showed the most potent activity (0.5 - 1 g/ml of MIC) against both MRSA and VRE. Royleanone exhibited weaker antimicrobial activity. The aromatic norditerpenoid 12-demethylmulticaulin was found to be active against a clinical isolated β-hemolytic Streptococcus (MIC of 2.0 µg/ml) while multicaulin and multiorthoquinone were particularly active against Staphylococcus aureus and β-hemolytic Streptococcus displaying a MIC of 0.2 and 0.1 µg/ml for both strains, respectively. Multicaulin was also active against Escherichia coli and Proteus mirabilis (MIC of 0.7 and 1.4 µg/ml, respectively) and multiorthoquinone against Enterococcus faecalis and Pseudomonas aeruginosa (MIC of 2.0 and 0.5 µg/ml, respectively) (Ulubelen et al. 1997). The antibacterial properties of cryptotanshinone and dihydrotanshinone I determined by the agar dilution method showed strong activity against a broad range of gram-positive bacteria (MIC values ranged from 3.1 to > 100 µg/ml), while they were less active against gram-negative bacteria (MIC values > 100 µg/ml). The authors suggest that the antibacterial action might resulted from superoxide radical generation, measured by the nitro blue tetrazolium (NBT) reduction method, even though they rule out the possibility that the permeability and/or binding properties to cell membranes cause the observed differences between gram-positive and -negative bacteria of these tanshinones (Lee et al. 1999b). A series of abietanes named hypargenins were screened against standard bacteria. Among them, hypargenin A and hypargenin B were found to be active against S. aureus and Klebsiella pneumoniae (MIC of 15.6 and 125 µg/ml, respectively) while hypargenin C showed activity against S. aureus and B. subtilis (MIC of 125 and 15.6 µg/ml, respectively). The abietane hypargenin D showed activity only against B. subtilis (MIC of 62.5 µg/ml) while hypargenin F was found to be active against S. aureus, S. epidermidis and P. aeruginosa (MIC of 125.0, 62.5 and 125 µg/ml, respectively) (Ulubelen et al. 1988). Moreover, tanshinones have been studied against Mycobacterium tuberculosis. Tuberculosis is an old human infectious disease that still remains a leading cause of death worldwide, not only in developing, but also in developed countries. M. tuberculosis is the most common mycobacterium causing human tuberculosis. Reports on natural products with anti-tuberculosis activity are increasing, and it is expected that these compounds will enlarge the diversity of novel scaffolds for the antimycobacterial drug development (Copp & Pearce 2007). The norabietanes multicaulin, 12-demethylmulticaulin, multiorthoquinone, 2- demethylmultiorthoquinone and the abietanes 12-methyl-5-dehydroacetylhorminone and 12- Introduction 13
methyl-5-dehydrohorminone were evaluated against M. tuberculosis strain H 37 Rv. All tested abietanes/norabietanes exhibited strong antituberculosis activity with MIC values of 5.6 µg/ml, 0.46 µg/ml, 2.0 µg/ml, 1.2 µg/ml, 0.89 µg/ml and 1.2 µg/ml, respectively (Ulubelen et al. 1997). The abietane diterpene hypargenin F, was also found to be active against M. tuberculosis (Ulubelen et al. 1988). Four natural royleanone abietanes and some hemisynthetic derivatives with the same chromophoric system were screened against the sensitive H 37 Rv and the multidrug-resistant (MDR) M. tuberculosis strain. The royleanones, which bear a p-benzoquinone C ring, i.e. 6 β,7 α-dihydroxyroyleanone, horminone and 6,7- dehydroroyleanone, showed mild activities against the MDR strain (MIC values ≤ 12.5 µg/ml). Distinctively, 7α-acetoxy-6β-hydroxyroyleanone exhibited potent antimycobacterial activity against the MDR strain with a MIC value of 3.12 µg/ml and against the H 37 Rv strain with a MIC value of 25 µg/ml. These results may suggest that the presence of the 7 α-acetoxy group at B ring is essential to increase the MDR M. tuberculosis activity (Rijo et al. 2010).
Antileishmanial activity The new abietane diterpenes 7-hydroxy-12-methoxy-20-nor-abieta-1,5(10),7,9,12-pentaen- 6,14-dione and 12-deoxy-royleanone were tested in vitro against extracellular promastigote and intracellular amastigote forms of Leishmania donovani , causative agent of visceral leishmaniasis, and Leishmania major , the causative agent of cutaneous leishmaniasis. The diterpenes showed appreciable in vitro antileishmanial activity against intracellular amastigote forms of both Leishmania donovani (IC 50 values of 170 and 120 nM, respectively) and
Leishmania major (IC 50 values of 290 and 180 nM, respectively). The standard therapeutic ® drug pentostam showed an IC 50 of 7.0 and 12.7 nM, respectively (Tan et al. 2002). Cryptotanshinone, 1 β-hydroxycryptotanshinone, 1-oxocryptotanshinone and 1-oxomiltirone also have demonstrated to be active against Leishmania major exhibiting in vitro antileishmanial activity with IC 50 values in the range of 18 to 47 µM (Sairafianpour et al. 2001).
Antifungal activity The rearranged abietane naphthoquinone sahandinone, its isomer and horminone were given to the growth media of four strains of two zygomycetes fungi, (+) and (-) Mucor mucedo and Blakeslea trispora . Only a slight inhibitory activity was observed at a dose of 100 14 Introduction
µg against (+) B. trispora (Jassbi et al. 2006a). 7-oxoroyleanone showed activity against Candida albicans with a MIC of 6.75 µg/ml (Ulubelen et al. 1994). Antifungal activities of twelve diterpenoids against two wood decay fungi, Trametes versicolor (white-rot) and Fomitopsis palustris (brown-rot) were studied by Kusumoto et al. 2010. 14-deoxycoleon U, taxodione and salvinolone were strong mycelium growth inhibitors in T. versicolor and antifungal activity against F. palustris. Taxodione and ferruginol also had antifungal activity against F. palustris . 6,7-dehydroferruginol, taxodal, and sugiol did not inhibit mycelial growth of T. versicolor but they reduced the growth rate of F. palustris . The results suggest that the position and the number of hydroxyl groups on abietane-type structures may be related to antifungal activities against T. versicolor and F. palustris .
Anti-inflammatory activity The common diterpenoid abietic acid was found to inhibit the soybean 5-lipoxygenase with an IC 50 of 29.5 ± 1.29 µM which may have beneficial effects in the treatment of a variety of diseases including allergy, asthma, arthritis and psoriasis (Ulusu et al. 2002). Abietic acid was also demonstrated to inhibit prostaglandin E2 (PGE 2) production in LPS-treated macrophages and significantly inhibited rat paw oedema induced by carrageenan in a time- and dose- dependent manner, and mouse ear oedema induced by 12-O-tetradecanoylphorbol acetate, after oral or topical administration (Fernandez et al. 2001). Abietic acid as well as its partial synthetically prepared derivatives abietic acid methyl ester and abietinol were tested for
COX-1, COX-2, and LTB 4 formation inhibitory activities. The diterpenes were mainly active in the LTB 4 formation assay. Abietinol was the most active compound exhibiting an IC 50 value of 5.9 µM. Abietic acid showed an IC 50 of 13.5 µM. The abietic acid methyl ester was virtually inactive (IC 50 > 125 µM) (Pferschy-Wenzig et al. 2008). The potential anti-inflammatory activity of sugiol and the relationship between signal transduction and inflammatory cytokines was evaluated in vitro by Chao et al. 2005. Sugiol exhibited effective inhibitory activity on proIL-1β, IL-1β and TNF-α production after LPS- stimulated macrophages in J774A.1 cells at 30 µM concentration and demonstrated to inhibit ERK1/2, JNK1/2, and p38 phosphorylation in LPS-stimulated macrophages in a range of 10 to 30 µM. The authors suggest that sugiol-mediated inhibition on TNF-α induced proIL-1β/ IL-1β protein expression and MAPKs phosphorylation may be, at least in part, due to the ROS scavenger activity. Introduction 15
Tanshinone I, cryptotanshinone and dihydrotanshinone significantly inhibit interleukin-12 (IL-12) and interferon-gamma (IFN-γ) production. Furthermore, it was found that tanshinones inhibit the expression of IL-12 p40 gene and nuclear factor (NF)-κB binding to the κB site, suggesting that tanshinones may negatively regulate IL-12 production at the transcription level (Kang et al. 2000). Tanshinone IIA was also found to inhibit the production of inflammatory mediators such as IL-1β, IL-6 and TNF-α (Jang et al. 2003).
Trypanocidal activity The trypanocidal activity of the abietane diterpenes 14-hydroxy-6-oxoferruginol, 14- hydroxy-6,12-dione-7,9(11),13-abietatriene, 14-hydroxy-12-oxo-7,9(11),13-abietatriene, 6,12,14-trihydroxyabieta-5,8,11,13-tetraen-7-one, ar-abietatriene-12-ol-6,7-dione-14,16- oxide, and ar-abietatriene-12,16-diol-14,16-oxide was described by Herrera et al. 2008. The cytotoxic activity was assayed against Trypanosoma cruzi epimastigote and trypomastigote forms. All bioactive compounds showed higher cytotoxicity against trypomastigotes, the infection form in the mammalian bloodstream, than against epimastigotes, the form present in the insect. The antitrypomastigote activity of the studied compounds were in the range of IC 50 ® 0.85 to 25 µM, respectively and were lower than the positive control benznidazole (IC 50 29.3 µM).
Antioxidant activity Nowadays, new developments in bio-medical science focus on free radicals which are involved in many diseases. There is increasing evidence that many degenerative diseases such as brain dysfunction, cancer and many others could be the result of cellular damage caused by free radicals. Therefore, antioxidants may play an important role in prevention and could also contribute to beneficial effects in therapeutic treatment of many diseases. The abietane-type diterpenoids, royleanone 12-methyl ether, 7-epi-salviviridinol, iguestol, ferruginol, taxodione, viridone and demethylinuroyleanol isolated from different Salvia species were studied for their antioxidant activity. The IC 50 values of the studied compounds using the ABTS assay showed that only taxodione and ferruginol exhibited significant antioxidant activity with IC 50 values of 5.39 and 2.37 µg/ml, respectively. The authors proposed that the antioxidant activity for taxodione may be explained by its enone moieties in B and C rings (Kolak et al. 2009). Studies on the scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals revealed that ferruginol possess a significant inhibitory activity, followed by hinokiol, secoabietane 16 Introduction dialdehyde, 6 β-acetoxy-7α-hydroxy-royleanone and isopimarinol, and with sugiol showing negligible radical scavenging activity. From these results it can be concluded that the phenolic structure present in the abietane-type diterpene is essential to its radical scavenging activity (Wang et al. 2002). Tanshinone I, tanshinone IIB, cryptotanshinone, dihydrotanshinone I, methylenetanshinquinone, miltirone I, dehydromiltirone and danshenxinkun B were found to act as antioxidants comparable to butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), whereas tanshinone IIA did not show antioxidant properties. It was speculated that tanshinones act as antioxidants by adding the lipid radical to the radical quinone to form a stabilized radical which interrupts the autoxidation chain reaction (Zhang et al. 1990; Weng & Gordon 1992). Tanshinone IIA (01 - 100 µM) demonstrated to be an effective antioxidant against LDL oxidation in vitro and the mechanism appears to be related to its peroxyl radical scavenging and LDL binding activities (Niu et al. 2000).
Cardiovascular activity For hundred of years S. miltiorrhiza extract has been widely used in China, Korea, Japan and other Asian countries because of its properties of improving microcirculation, causing coronary vasodilatation, suppressing the formation of thromboxane, inhibiting platelet adhesion and aggregation, and protecting against myocardial ischemia (Cheng 2007). The crude extract and the isolated compounds from three different Salvia species ( S. amplexicaulis, S. eriophora and S. syriaca ) were investigated in vivo on Wistar Albino rats for the cardiovascular properties. The crude extract of S. eriophora and eleven isolated diterpenoids were tested for cardiovascular activity. Among the tested abietanes, especially ferruginol, aethiopinone and 4,12-dihydroxysapriparaquinone as well as the crude extract showed antihypertensive activity by significantly reducing arterial blood pressure. This activity was considered probably due to the vasorelaxation activity (Ulubelen et al. 2002). Diterpenes isolated from S. syriaca and S. amplexicaulis were also tested for cardioactivity. Ferruginol, 3 β-hydroxystigmast-5-en-7-one and 7-oxo-abieta-9,12,14-triene were found to be active in significant reduction of blood pressure, without influencing the heart rate, in the same range as the positive controls regitine and propranolol (Ulubelen et al. 2000; Kolak et al. 2001).
Introduction 17
Cytotoxic and antitumor activities Cytotoxic activity is one of the most investigated activities of diterpenes, especially in tanshinones using different models and cell lines. The in vitro antiproliferative activities of crude extracts from different Salvia species ( S. dominica , S. menthaefolia , S. palaestina , S. sclarea, S. spinosa, S. fruticosa, S. pomifera, S. ringens and S. verbenaca ), which are known to have a high contents of diterpenes, were screened against different human cancer cell lines. All tested extracts showed some degree of cytotoxicity dependent on the cell line studied with an IC 50 values ranging from 42 to 400 g/ml (Fiore et al. 2006; Badisa et al. 2004). These data suggested that there are great differences between the various species. Nevertheless, the results demonstrate that the genus Salvia can be considered as a natural source of potential antitumor agents. The cytotoxic activity of eight abietane diterpenes was evaluated in vitro against five different cancer cell lines (ovarian cancer, non-small-cell lung cancer, colon cancer and two breast cancer cells). Growth inhibition 50% (GI 50 ) values were determined and the majority of the compounds assayed showed antiproliferative activity against sensitive and resistant tumor cell lines. According to the obtained results, the most active compounds (carnosol, 20- deoxocarnosol and 16-hydroxycarnosol) showed GI 50 values in the range of 3.6 - 5.4 µM against A2780 ovarian and HBL-100 breast cell lines (Guerrero et al. 2006).
Ten diterpenoids (6 α-hydroxysalvinolone, 6 α-hydroxytaxodone, aethiopinone, microstegiol, ferruginol, saprorthoquinone, 11,12-dioxoabieta-8,13-diene, taxodione, hypargenin A and hypargenin D) were isolated from the roots of Salvia hypargeia and tested against a panel of human cancer cell lines like human breast cancer (BC 1), human lung cancer (LU 2), human colon cancer (Col 2), human epidermoidal carcinoma in mouth (KB), vinblastine-resistant KB-VI, hormone-dependent human prostate cancer (LNCaP) as well as
P388 and ASK cells. Taxodione was the most active substance with an ED 50 from 0.3 to 5.1 µg/ml in all tested cell lines. 6 α-hydroxytaxodione and ferruginol showed selective activity against colon cancer cells with an ED 50 of 9 and 3.3 µg/ml and for human prostate cancer cells with an ED 50 of 12.9 and >12 µg/ml, respectively. The other compounds were either not active or exhibited only weak cytotoxicity. The activity of all compounds in the multidrug-resistant KB-VI cell culture system was neglectable (Ulubelen et al. 1999b). Cytotoxic activity against HeLa (human cervical cancer cells) and Hep-2 (human laryngeal carcinoma cells) cell lines was studied using eight abietane diterpenes isolated from S. mellifera by Moujir et al. 1996. Ferruginol, taxodione, isotanshinone III and 7 α- 18 Introduction hydroxyroyleanone exhibited cytotoxic activity against both HeLa and Hep-2 cells in culture.
7 α-hydroxyroyleanone was the most cytotoxic compound with an IC 50 of 13.5 and 15.8
µg/ml, respectively. Royleanone was only moderate active on Hep-2 cells (IC 50 34 µg/ml). The diterpenes bearing a p-quinone moiety, such as, 6,7-dehydroroyleanone, royleanone, 7,20-epoxyroyleanone and horminone as well as the phenolic diterpene 7-oxo-11,12,14- trihydroxy-8,11,13-abietatrien-20-al isolated from Sphacele chamaedryoides showed cytotoxic activity (IC 50 values of 61, 59, 57, 12 and 43 µM, respectively) against gastric adenomatous carcinoma (AGS) cell, with horminone being the most active (Areche et al. 2009).
The cytotoxicity of sugiol (IC 50 from 8 to >100µM) (Costa-Lotufo et al. 2004; Jonathan et al. 1989; Son et al. 2005; Kupchan et al. 1968; Iwamoto et al. 2003), horminone (IC 50 values from 12 - 40 µM) (Areche et al. 2009; Jonathan et al. 1989), royleanone (IC 50 values from 34 -
80 µM) (Areche et al. 2009; Kupchan et al. 1968), cryptojaponol (IC 50 34 µM) (Topcu et al.
2003), ferruginol (IC 50 values from 3 - 33 µM) (Topcu et al. 2003; Son et al. 2005), 7 α- acetoxyroyleanone (IC 50 values from 2.5 - 39 µM) (Araujo et al. 2006; Slamenova et al. 2004) and many others abietane diterpenes have been investigated over the years and exhibited some degree of cytotoxicity depending on the tested cell line. Tanshinones are considered the most cytotoxic abietane diterpenes. A cytotoxic-guided fractionation of the roots of S. miltiorrhiza afforded 18 tanshinones. Antiproliferative activity against five cancer cell lines demonstrated that all tested compounds exhibited a significant but presumably nonspecific cytotoxicity against all examined tumor cells (IC 50 values ranged from 0.2 to 8.1 µg/ml). It was suggested that the cytotoxicity of tanshinones against tumor cells might be due to the common naphthoquinone skeleton rather than to the substituent attached to it (Ryu et al. 1997). Tanshen, the rhizome of S. miltiorrhiza Bunge, is one of the most important ancient Chinese herbal drugs. Chemical and biological studies have continued to increase and provide a scientific basis for the traditional clinical use of Tanshen and also contributed to the development of new drug candidates. Currently, nearly 40 variants of the basic tanshinone structure have been found in the roots of S. miltiorrhiza and 32 compounds have been tested for cytotoxicity against several human tumor cell lines. In general, the tanshinones with an ortho -quinone and an intact ring D exhibited significant cytotoxicity (IC 50 values in a range of 0.4 to 4.0 µg/ml), while compounds with a para -quinone and/or without ring D, had only Introduction 19
weak or no cytotoxicity (IC 50 values > 4 µg/ml) (Wang et al. 2007; Wang 2010; Wu et al. 1991). Altogether, many papers have described the isolation and the preliminary cytotoxic studies of abietane diterpenes against different human cancer cell lines. The majority of these abietanes have shown only moderate activity. However, some diterpenes, especially the tanshinones, exhibited interesting and promising antiproliferative and cytotoxic effects. Various studies which further explore and discuss the cytotoxic activity of these small- molecules have also been described, but the exact molecular mechanism helpful for a therapeutic basis remains poorly understood.
The diterpenes royleanone, horminone and 7 α-acetoxyroyleanone were demonstrated to decrease the viability of human colon carcinoma and hepatoma cells proportionally to the concentration and time of treatment. Cytotoxicity did not correlate with induction of apoptosis, however all diterpenes tested at 0.1 µM induced DNA damage evaluated by alkaline elution technique and the comet assay. The authors suggested that the DNA strand breaks detected was originated from alkali-labile lesions and not as a consequence of an enzymatic ( i.e. topoisomerase) action or as a result of oxidative radical attack (Slamenova et al. 2004).
Araujo et al. 2006 studied the antiproliferative effects of 7 α-acetoxyroyleanone in five tumor cell lines. The IC 50 values ranged from 2.5 µM in skin cancer cells to 21.1 µM in colon cancer cells. Typical morphological changes which are characteristic for apoptosis, such as chromatin condensation and fragmentation of the nuclei were observed in 27% of the cell population after 10 µg/ml treatment for 24h. No necrotic cells were detected. The cytotoxic activity against a panel of ten cancer cell lines and the effects on topoisomerase I for 324 natural compounds were screened by Han et al. 2008. The abietane type diterpene 7-ketoroyleanone showed cytotoxic activity against nine cell lines with IC 50 values ranging from 13.14 to 25.67 µM. Cryptotanshinone and dihydrotanshinone I display cytotoxicity against 4 and 5 tested cell lines, and were more active against non-small lung carcinoma cells (IC 50 values of 6.31 and 5.46 µM, respectively). 7-ketoroyleanone, cryptotanshinone and dihydrotanshinone I also inhibited human DNA topoisomerase I in the relaxation assay (IC 50 values of 38.9, 22.7 and 66.8 µM, respectively). Spiridonov et al. 2003 suggested that the biological membranes may be a target of royleanones due to their lipophilic character and the protonophoric activity may contribute to the cytotoxic mechanism of action of these compounds. Royleanone, 7-ketoroylenanone and 20 Introduction
7α-acetoxyroyleanone (at 3 µg/ml) showed proton conductivity in artificial lipid membranes, uncoupling action on mitochondrial respiration and phosphorylation in vitro. Moreover, cytotoxic activity on human lymphoblastoid Raji cells was observed. The abietane diterpene 6-hydroxy-5,6-dehydrosugiol (HDHS) showed significant reduction of cell viability at a 10 µM concentration in a LNCaP, an androgen-dependent prostatic cancer cell line after 48h treatment. A significant increase in the sub-G1 cell population along with decreases in the G0/G1 and S phase was observed after 48h HDHS treatment. Induction of apoptosis, cleavage of poly (ADP-ribose) polymerase (PARP), activation of caspase-3 and caspase-7, up-regulation of p53 expression and decrease of antiapoptotic Bcl-xL was observed after 5 to 10 µM treatment of HDHS for 24h. The molecular mechanism for HDHS-induced cell cycle arrest was correlated with a concentration-dependent decrease in the protein expression of cyclins (D1 and E) and cyclin-dependent kinases (CDK4, CDK6, and CDK2). Moreover, an increase was observed in protein expression of CDK inhibitors p21 and p27 and phosphorylation of retinoblastoma. In vivo experiments using athymic nude mice with 22Rv1 tumor revealed a decrease in tumor growth of about 22% and 39%, respectively, without exhibiting any symptoms of toxicity after 24-days of daily administration of 0.5 and 2.5 mg/kg body weight (Lin et al. 2008). Ferruginol exhibited cytotoxic effects against an androgen-independent human prostate cancer cell line (PC3) (IC 50 value of 55 µM) via activation of caspase-8, -9 and -3 as well as apoptosis-inducing factor (AIF). 15% and 30% of apoptotic nuclei was detected after 25 and 50 µM of ferruginol treatment whereas the ratio Bcl2:Bax was not affected. Ferruginol downregulated the p85 subunit of PI3K at a concentration of 50 µM, decreased the phosphorylation levels of STAT3 and STAT5, activated the MAPK p38, and slightly inhibited ERK2. Cell cycle arrest at G0/G1 was correlated with increased expression of p21 and decreased levels of CDK4, CDK6, cyclin D1 and cyclin D3 (de Jesus et al. 2008). Salvicine, a structurally modified diterpenoid quinone derived from S. prionitis is currently under Phase II clinical trials for cancer therapy. It displays potent antitumor activities in vitro against a panel of cancer cell lines especially against lung (mean IC 50 13.02 µM) and gastric cancer cells (mean IC 50 9.25 µM). Salvicine (12.5 µM) exhibited perturbation of cell cycle progression and increased the proportion of cells in the G1 phase from 44.8% to 69.29%, followed by a decrease in the S phase from 45.73% to 14.22%. Only a slight change was observed in the G2/M phase (Qing et al. 1999). Salvicine effectively kills multidrug-resistant (MDR) sublines, such as K562/A02, KB/VCR and MCF- 7/ADR, and parental K562, KB and Introduction 21
MCF-7 cell lines to an equivalent degree. These cytotoxic activities were much more potent than those of classical anticancer drugs (average resistance factor: 1.42 for salvicine vs. 233.19 and 71.22 for doxorubicin and etoposide, respectively). The anticancer activity of salvicine was associated with its ability to induce apoptosis. The compound activated caspase- 1 and -3 (but not caspase-8) and increased the ratio of bax to bcl-2 mRNA via reduction of bcl-2 mRNA expression. Furthermore, salvicine induced the downregulation of mdr-1 gene and P-gp expression in MDR K562/A02 cells. These results suggested that the reduction of mdr-1 and bcl-2 expression by salvicine possibly contributes to its cytotoxicity and apoptotic induction (Miao et al. 2003). Salvicine acted as a topoisomerase II (topo II) poison through its marked enhancement effect on DNA double-strand breaks observed in the decatenation assay
(IC 50 value of 3 µM). In contrast, no inhibitory activity was observed against the catalytic activity of topo I, suggesting a highly selective effect on topo II. Salvicine was classified as a novel non-intercalative topo II poison which induces protein-linked DNA breaks (Meng & Ding 2007). The possible binding sites on human topo II α and the molecular interactions were further investigated. Molecular modeling studies predicted that salvicine binds to the ATP pocket in the ATPase domain and superimposes on the phosphate and ribose groups. Salvicine functioned as an ATP competitor and exhibited activity distinct from that of other classical topo II agents (Hu et al. 2006). Moreover, Cai et al. 2007 further demonstrated that salvicine formed reactive oxygen species (ROS) which contributed to its induction of cytotoxicity in MDR cells, DNA double strand breaks and induction of apoptosis. Tanshinone I and tanshinone IIA displayed cytotoxicity at concentrations of 8 - 90 µM and 64 - 230 µM, respectively, depending on the cell line (A549, K562 and Colo205). Both suppressed AP-1 function and reduced the interaction of jun-fos and DNA with IC 50 values of 0.15 and 0.22 µM, respectively, in TPA induced NIH3T3 cells (Park et al. 1999). The anticancer effects of tanshinone I on the highly invasive human lung adenocarcinoma cell line, CL1-5 was studied by Lee et al. 2008. Tanshinone I significantly inhibited migration, invasion and gelatinase activity in macrophage-conditioned medium-stimulated CL1-5 cells in vitro and also reduced the tumorigenesis and etastasis in CL1-5-bearing severe combined immuno-deficient mice. These effects were mediated at least partly through interleukin-8, Ras -mitogen-activated protein kinase, and Rac 1 signaling pathways. Tanshinone I exhibited a dose-dependent reduction on cell viability on rat hepatic stellate cells (T-HSC/Cl-6) after 24h exposure at 0.5 - 25 µM concentrations. Treatment of T-HSC/Cl- 6 cells with 5 and 10 µM of tanshinone I for 24h resulted in induction of apoptosis which was 22 Introduction characterized by increasing caspase-3 activity, cytochrome c release and loss of mitochondrial membrane potential (Kim et al. 2003). Later on Nizamutdinova et al. 2008b investigated the effect of tanshinone I at 1 - 50 µM concentration on the induction of apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) in vitro . Tanshinone I inhibited the cell proliferation of MCF-7 and MDA-MB-231 cells in a dose and time-dependent manner. At 50 µM, tanshinone I increased the apoptotic cell population in MCF-7 cells to 14, 26 and 27% after 24, 48 and 72h of incubation, respectively. The induced apoptosis by tanshinone I in the above mentioned two cell lines was via activation of caspase-3, downregulation of the anti- apoptotic protein Bcl-2 and up-regulation of the pro-apoptotic protein Bax . Similar results were obtained from Su et al. 2008. They studied the ability of tanshinone I to induce apoptosis in human colon cancer Colo 205 cells. When these cells were treated with tanshinone I at 0, 1, 2.5, 5 and 10 g/ml for 72h, the percentage of cells in the sub-G1 phase increased from 3.83% to 7.22%, 8.68%, 14.4% and 32.98%, respectively. Their results also demonstrated that treatment with 2.5 and 5 µg/ml for 24h increased activation of p53 , p21 , bax and caspase-3. The authors suggested that tanshinone I induced apoptosis in Colo 205 cells through both mitochondrial-mediated intrinsic cell-death pathways and p21 -mediated G0/G1 cell cycle arrest. Tanshinone I showed mild cytotoxicity in human umbilical vein endothelial cells (HUVECs) at the concentrations of 1 - 50 µM after 24h. Additionally, tanshinone I completely suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α stimulated HUVECs in a dose dependent-manner (1 - 50 µM). Tanshinone I also reduced adhesion of monocyte U937 and MDA-MB-231 cells to HUVECs. Moreover, it displayed inhibitory activity against TNF-α-induced production of vascular endothelial growth factor (VEGF) and VEGF-mediated tube formation in HUVECs. At 10 and 50 µM, tanshinone I significantly inhibited (> 80%) invasion of cancer cells. In addition, in nude mice treated with tanshinone I, both tumor mass volume and metastasis of MDA-MB-231 breast cancer cells to lung tissue markedly decreased (Nizamutdinova et al. 2008a). More recently, tanshinone I was reported to inhibit the proliferation of three different monocytic leukemia cells (U937, THP-1 and SHI 1) at doses of 10 to 50 µg/ml. Tanshinone I reduced the expression of hTERT mRNA, as well as the activity of telomerase, and down- regulated survivin levels. After 48h treatment with 10 - 50 µg/ml tanshinone I increased caspase-3 activity and cleavage of PARP. The authors suggested that the induction of Introduction 23 apoptosis by tanshinone I in monocytic leukemia U937 THP-1 and SHI 1 cells was highly correlated with activation of caspase-3 and decrease in telomerase activity as well as in mRNA expression of hTERT and survivin (Liu et al. 2010). Tanshinone I, together with dihydrotanshinone, also showed potent growth-inhibitory effects against various human hepatocellular carcinoma (HCC) cell lines including Hep3B and R-HepG2 which possess a mutant p53 oncogene and Pgp (p-Glycoprotein) overexpression, respectively. Both proteins are known to lead to the development of multidrug resistance. Induction of apoptotic cell death by tanshinone I (at 25 µM) and dihydrotanshinone (6.25 µM) were correlated with poly(ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3. The interaction of tanshinone I (6.25 µM) with doxorubicin on the growth inhibition of R-HepG2 cells enhanced the cytotostatic effect of doxorubicin at about 50% evaluated by the Combination Index. The authors observed that the cytotoxic effect of these compounds was reduced in the presence of p53 deficiency or mutation and Pgp overexpression. However, the growth- inhibitory effects were still greater than that produced by doxorubicin (Lee et al. 2010b). Hence, different targets have been addressed for the abietane diterpenes, further studies are necessary to enhance our knowledge on the cellular and molecular mechanisms of these interesting small molecules before selecting a lead for the development of anticancer drugs.
24 Introduction
1.4. Aim of the thesis
As mentioned above a lot of traditional medicinal plants are used for the treatment of various diseases, often without knowledge on the effective compounds. In this thesis, phytochemical and biological investigations were performed to identify and characterize the secondary metabolites in the roots of Peltodon longipes which are used as an antiseptic and anti-inflammatory remedy. Isolation of the main compounds was carried out using different chromatographic techniques such as thin liquid chromatography, open column chromatography, and low and high pressure liquid chromatography. Structure elucidation of the compounds was based on 1D and 2D NMR data ( 1H and 13 C, COSY, HSQC and HMBC), EI-MS and UV/VIS spectroscopic methods. Detailed studies were undertaken with the isolated compounds to clarify their cytotoxic molecular mechanism. Moreover, a quantitative cell-based assay was established to gain first insights in the wound healing properties of medicinal plants. Several plant extracts prepared from Brazilian medicinal plants were screened in this assay in addition to their effects on elastase.
Results 25
2. Results
2.1. Scratch assay
The scratch assay is a reliable in vitro method to get first insights into the wound healing properties of a compound or a plant extract. When keratinocytes are used, re-epithelialization processes can be studied, whereas the effect on granulation tissue can be analyzed with fibroblasts (Gurtner et al. 2008). Several papers describe the performance of the scratch assay (Liang et al. 2007; Phan et al. 2001; Yarrow et al. 2004). Here, the scratch assay was optimized to be suitable for the first evaluation on the rebuilding of new granulation tissue, which means for the quantification of fibroblast migration to and proliferation into the wounded area.
Establishment and improvement of the scratch assay To evaluate the effect on the wound repair of an extract or compound and to have a quality control for the assay, the use of a standard is required. A number of growth factors and cytokines have been reported to affect fibroblast motility directly or indirectly. As the role of platelet-derived growth factor (PDGF) in wound healing is well characterized, PDGF was taken as a positive control (Hosang et al. 1989; Clark et al. 2007). To find out the best concentration for the positive control, murine 3T3 fibroblasts were stimulated with PDGF at a concentration of 0.5 to 15 ng/ml. Quantification was done by staining of cell nuclei with 4',6- diamino-2-phenylindole (DAPI). Photographs were taken in a fluorescent microscope and analyzed using CellC software (Selinummi et al. 2005). Low concentrations of 0.5, 1 and 2 ng/ml resulted in a good correlation between dosage and an increased number of fibroblasts in the denuded area compared to the control (Figure 2.1 and Figure 2.2). In contrast, higher concentrations of 4 and 15 ng/ml induced lesser migration. Therefore, 2 ng/ml of PDGF was selected as positive control. 26 Results
(A) (B) (C) (D)
Figure 2.1 - Fluorescent microscope images to evaluate wound healing in vitro in the scratch assay using a confluent monolayer of 3T3 fibroblasts. Cell migration and proliferation into the wound was observed in response to an artificial injury. A single representative area is shown immediately after the wounding (A), a control group (B), after treatment with 2 ng/ml PDGF (C), and after treatment of 10 µg/ml of an n-hexane extract of Calendula officinalis (D) after 12h of incubation.
80
70 66.3
60
47.5 50
40 33.5
30 %cell numbers
20 17.2 10.9 10
0 0.5 ng/ml 1 ng/ml 2 ng/ml 4 ng/ml 15 ng/ml PDGF concentration
Figure 2.2 - Effect of 0.5, 1, 2, 4 and 15 ng/ml of PDGF after 12h of incubation (37 ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS on confluent monolayer cultures of fibroblasts. Data are expressed as percentage of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments.
To elucidate the optimal treatment time, cellular proliferation and migration of fibroblasts were studied after 8, 10, 12 and 14h of incubation using 2 ng/ml of PDGF. An incubation time of 14 hours resulted in the highest number of migrated cells in the denuded area (Figure 2.3). However, density of the cells was too high to be counted and distinguished unambiguously due to cluster building. A 12-hour incubation time allowed an accurate measurement. Results 27
80
70 66.4
60 51.2 50 46.7
40
30 % cell numbers %cell 20.6 20
10
0 8h 10h 12h 14h Incubation time (hours)
Figure 2.3 - Effect of 2 ng/ml of PDGF after 8, 10, 12 and 14h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS on confluent monolayer cultures of 3T3 fibroblasts. Data are expressed as percent of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments.
DMSO was used as a solvent for the preparation of stock solutions from the extracts and isolated compounds. Studies with different concentrations of DMSO (0.25% to 1.5%) showed toxic effects at concentrations above 1%, whereas lower concentrations (< 0.5%) of DMSO exhibited negligible effects. Therefore only DMSO concentrations smaller than 0.5% were used in further experiments.
Studies on European traditional plants, single compounds and pharmaceutical preparations The effect of two different preparations, a n-hexane and an ethanolic extract, from Calendula officinalis flowers, were investigated on fibroblasts migration and proliferation under the established conditions. As showed in Figure 2.4, the n-hexane extract of Calendula increased cell numbers to 54.8% ± 1.59 and 64.4% ± 1.60 at 1 and 10 µg/ml concentrations. The same concentrations of the ethanolic extract gave slightly higher cell numbers of 60.8% ± 4.36 and 70.5% ± 2.64, respectively. Comparing the results with the control group, a significant difference ( p<0.05) was observed in all tested concentrations. The activity of the Calendula extracts in promoting wound healing was similar to that of PDGF at a concentration of 2 ng/ml. 28 Results
The n-hexane and ethanolic extracts from Matricaria recutita flowers were also investigated under the same conditions (Figure 2.4), resulting in a much lower migration and proliferation rate with values of 13.1% ± 0.18 and 25.7% ± 0.86 for 1 and 10 µg/ml of the n- hexane extract and 6.4% ± 1.67 and 24.4% ± 0.60 for the respective concentrations of the ethanolic extract. The changes were not statistically significant ( p<0.05). Analysis of a commercial pharmaceutical preparation of Hypericum perforatum oil was also carried out (Figure 2.4). Only a concentration of 0.5 µg/ml was studied and gave a higher cell number of 19.9% ± 1.05 (not significant ( p<0.05). Higher concentrations were cytotoxic.
80 70.5 70 64.4 63.2 60.8 60 54.8
50
40
% cell numbers % cell 30 25.7 24.4 19.9 20 13.1 10 6.4
0 1 2 3 4 5 6 7 8 910
Figure 2.4 - Effect of preparations from Calendula officinalis , Matricaria recutita and Hypericum perforatum oil on the migratory and proliferative activities of fibroblasts in the scratch assay after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS. 1: PDGF (2 ng/ml); 2 and 3: n-hexane extract of Calendula officinalis (1 and 10 µg/ml); 4 and 5: ethanolic extract of Calendula officinalis ; 6 and 7: n-hexane extract of Matricaria recutita (1 and 10 µg/ml); 8 and 9: ethanolic extract of Matricaria recutita , (1 and 10 µg/ml); 10: Hypericum perforatum oil (0.5 µg/ml). Data are expressed as percentage of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments.
The extracts of Calendula increase the population of fibroblasts in the scratched area due to immigration of cells and/or proliferation of the migrated cells. To differentiate between these two distinct processes, mitomycin C (5 µg/ml) was applied to the wounded monolayer cultures of fibroblasts together with either PDGF (2 ng/ml) or, as example, the n-hexane extract from Calendula (1 and 10 µg/ml). Addition of mitomycin C blocks mitosis and allows discriminating between stimulation of migration and proliferation (Schreier et al. 1993). The cell numbers decreased to about 41.1% and 46.5%, respectively, when Calendula extracts Results 29 were applied, and a similar decrease to 47.1% was also observed with PDGF (see Figure 2.5, bars 1 - 3).
60 51.4 50 46.5 47.1 45.5 41.1 40
29.4 30 26.1 % cell numbers %cell
20
10
0 1 2 3 4 5 6 7
Figure 2.5 - Effect of PDGF, n-hexane extract of Calendula officinalis and triterpene esters on the migratory activities of fibroblasts in the scratch assay in the presence of 5 µg/ml of antimitotic mitomycin C after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS. 1: PDGF (2 ng/ml); 2 and 3: n-hexane extract of Calendula officinalis (1 and 10 µg/ml); 4 and 5: faradiol myristate (10 and 50 µg/ml); 6 and 7: faradiol palmitate (10 and 50 µg/ml). Data are expressed as percentage of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments.
One of the active components of Calendula which are thought to be responsible for its anti-inflammatory activity, are triterpenoids, in particular faradiol monoesters. To investigate whether the triterpene monoesters from Calendula do not only possess an anti-inflammatory activity, but also stimulate fibroblast proliferation and migration, faradiol palmitate and faradiol myristate were studied under the same conditions. When tested at 10 µg/ml (15.31 µM) and 50 µg/ml (76.55 µM) faradiol myristate showed an increase in cell number of 37.9% ± 2.32 and 73.3% ± 2.43, respectively. Similarly, faradiol palmitate increased the number of fibroblasts to 47.5% ± 2.66 and 67.5% ± 2.07 in the scratched area when tested at the same concentration. The difference was significant when compared to the control ( p<0.05) (see Figure 2.6). Again, studies were carried out to determine to which extent proliferation contributed to the enhancement of fibroblasts in the denuded area after the scratch. Combined incubation of the antimitotic mitomycin C (5 µg/ml) with faradiol myristate or faradiol palmitate (10 and 50 µg/ml, respectively) revealed a similar decrease in cell numbers as observed for the n-hexane extract of Calendula (see Figure 2.5). 30 Results
80 73.3 67.5 70 65.1 60
50 47.5
40 37.9
% cell numbers %cell 30
20
10
0 1 2 3 4 5
Figure 2.6 - Effect of the triterpene esters faradiol myristate and faradiol palmitate on the migratory and proliferative activities of fibroblasts in the scratch assay after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS. 1: PDGF (2 ng/ml); 2 and 3: faradiol myristate (10 and 50 µg/ml); 4 and 5: faradiol palmitate (10 and 50 µg/ml). Data are expressed as percentage of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments.
Studies of Brazilian plant extracts The lipophilic and hydrophilic extracts from twelve plants which are used as wound healing agents in traditional South Brazilian medicine were study in the scratch assay to evaluate the influence on fibroblasts migration to and proliferation into the wounded monolayer. It was not differentiated between migration and proliferation. All extracts were studied at a 10 µg/ml concentration and the stimulation rate was expressed in percentage (see Table 2.1). Both extracts from Waltheria douradinha turned out to possess the highest activity to move in and fill the damaged area after the physical injury ( 11E : 54.7% and 11H : 79.7%), next to Schinus molle ( 9E : 76.2% and 9H : 50.8%) and Galinsoga parviflora ( 3E : 59.8% and 3H : 64.3%). Other extracts exhibited a moderate migration activity, such as Pluchea sagittalis (7E : 43.9% and 7H : 40.7%), Iresine herbstii ( 4E : 34.3% and 4H : 28.3%) and Sedum dendroideum ( 10E : 27.9% and 10H : 37.2%). Otherwise, some extracts as Eupatorium laevigatum , Kalanchöe tubiflora and Xanthium cavanillesii showed a moderate “wound healing” effect only for the ethanolic extract ( 2E : 30.1%, 5E : 46.2% and 12E : 41.2%).
Results 31
Table 2.1 - Effects of Brazilian plant extracts at 10 µg/ml concentration on cellular migration and proliferation evaluated in the scratch assay. Plant Extract Migration/Proliferation activity (%) n-Hex – H 26.6 ± 3.2* Brugmansia suaveolens (1) EtOH – E 9.8 ± 3.2 n-Hex – H -13.0 ± 2.7 Eupatorium laevigatum (2) EtOH – E 30.1 ± 3.2* n-Hex – H 64.3 ± 1.2* Galinsoga parviflora (3) EtOH – E 59.8 ± 2.4* n-Hex – H 28.3 ± 2.4* Iresine herbstii Hook. (4) EtOH – E 34.3 ± 2.9* n-Hex – H 4.7 ± 2.0 Kalanchöe tubiflora (5) EtOH – E 46.2 ± 4.2* n-Hex – H 1.7 ± 1.5 Petiveria alliacea L (6) EtOH – E 10.3 ± 2.0 n-Hex – H 40.7 ± 2.5* Pluchea sagittalis (7) EtOH – E 43.9 ± 1.4* n-Hex – H -35.6 ± 3.6* Piper regnellii (8) EtOH – E 22.1 ± 2.2* n-Hex – H 50.8 ± 3.1* Schinus mole L. (9) EtOH – E 76.2 ± 2.8* n-Hex – H 37.2 ± 0.1* Sedum dendroideum (10) EtOH – E 27.9 ± 0.8* n-Hex – H 79.7 ± 0.8* Waltheria douradinha (11 ) EtOH – E 54.7 ± 1.7* n-Hex – H 9.9 ± 2.6 Xanthium cavanillesii (12) EtOH – E 41.2 ± 1.8* Data represent the mean ± S.E.M. of two independent experiments. Significant differences between the treated groups and the control was determined with the Student’s t-test at a level of * p < 0.05.
32 Results
2.2. Studies of Brazilian medicinal plants on HNE
To support the traditional use as wound healing remedy several bioassays with the lipophilic and hydrophilic extracts from twelve plants were performed. The extracts were studied for their effect on human neutrophil elastase release and elastase activity as described in section 5.2.3.2. All extracts were studied at 10, 50 and 100 µg/ml concentration and the results on release and/or direct inhibition on elastase were expressed in percentage. The extracts that show similar values in the elastase inhibition and the elastase release assay, were considered to have no effect on elastase release. As showed in Table 2.2, three groups could be differentiated: the extracts from one group showed no or only a neglectable activity, like 2H , 3H , 3E , 6H , 10H and 10E . In the other group the extracts were put together which had a dominating inhibitory effect on elastase release, such as 2E , 4E , 7E , 8E , 11H , 12H and 12E . An additional direct inhibitory effect on elastase cannot be excluded. The extract 8H was special, once activity on elastase release decreased with increasing concentrations. Extracts from the third group, 1H , 1E , 4H , 5H , 5E , 6E , 7H , 9H , 9E and 11E , mainly directly impair elastase activity. Again, slight effects on its release maybe occur. In summary, most of the extracts have an effect on elastase which may have beneficial effects on skin elasticity and inflammation.
Results 33
Table 2.2 - Results from the elastase assay using the n-hexane and the ethanolic extracts of twelve Brazilian medicinal plants. Human Neutrophil Elastase (%) Name of the Extract Release Direct inhibition plant species 10 µg/ml 50 µg/ml 100 µg/ml 10 µg/ml 50 µg/ml 100 µg/ml
Brugmansia n-Hex – H -18.9 ± 0.9 -7.7 ± 0.8 36.9 ± 2.4 24.5 ± 1.9 43.1 ± 1.5 67.0 ± 0.3 suaveolens (1) EtOH – E 9.2 ± 1.8 55.6 ± 2.3 65.9 ± 1.8 20.8 ± 0.9 51.7 ± 0.8 51.3 ± 0.6
Eupatorium n-Hex – H -7.5 ± 1.6 -6.3 ± 0.4 23.3 ± 1.8 27.8 ± 1.2 38.2 ± 2.1 44.0 ± 1.0 laevigatum (2) EtOH – E 16.1 ± 2.4 67.6 ± 1.6 86.2 ± 1.3 5.3 ± 1.0 29.6 ± 2.1 48.8 ± 1.2
Galinsoga n-Hex – H -7.6 ± 0.8 11.6 ± 1.7 35.8 ± 0.7 11.5 ± 1.9 19.9 ± 1.5 36.6 ± 2.4 parviflora (3) EtOH – E -1.6 ± 0.6 33.6 ± 1.8 31.1 ± 1.7 -0.1 ± 0.3 18.2 ± 0.4 34.5 ± 0.3
Iresine herbstii n-Hex – H 2.0 ± 0.6 23.3 ± 0.6 39.2 ± 0.7 10.3 ± 0.5 44.7 ± 0.6 59.6 ± 0.6 Hook. (4) EtOH – E 6.6 ± 0.8 24.1 ± 0.7 42.6 ± 1.6 6.4 ± 0.8 16.5 ± 1.6 13.0 ± 0.9
Kalanchöe n-Hex – H -3.9 ± 0.7 16.9 ± 0.4 39.4 ± 0.8 28.6 ± 0.6 69.6 ± 0.5 85.9 ± 0.5 tubiflora (5) EtOH – E -2.3 ± 1.1 21.9 ± 1.7 43.2 ± 1.0 11.7 ± 1.0 53.6 ± 0.6 74.7 ± 0.8
Petiveria n-Hex – H -7.1 ± 0.9 -5.0 ± 0.7 17.3 ± 1.0 0.5 ± 1.5 25.8 ± 1.1 51.5 ± 0.9 alliacea L (6) EtOH – E 29.5 ± 1.1 58.1 ± 0.9 54.4 ± 1.7 26.7 ± 0.4 67.5 ± 0.7 66.6 ± 1.3
Pluchea n-Hex – H 18.4 ± 1.2 33.9 ± 1.4 40.3 ± 1.1 56.5 ± 1.1 60.5 ± 1.1 61.6 ± 0.6 sagittalis (7) EtOH – E 30.2 ± 1.4 81.8 ± 1.6 82.9 ± 0.6 23.0 ± 1.2 65.7 ± 0.2 75.3 ± 0.9
Piper regnellii n-Hex – H 46.3 ± 2.2 27.1 ± 1.2 15.6 ± 1.1 3.5 ± 1.9 -19.6 ± 1.5 -39.0 ± 1.6 (8) EtOH – E 61.9 ± 1.0 77.7 ± 1.4 78.7 ± 1.5 37.1 ± 1.5 35.5 ± 1.3 27.3 ± 1.7
Schinus mole n-Hex – H -19.9 ± 1.6 22.6 ± 1.1 66.9 ± 2.3 47.1 ± 1.4 59.8 ± 0.4 64.3 ± 2.4 L. (9) EtOH – E 14.4 ± 1.2 54.7 ± 1.4 74.0 ± 0.8 19.8 ± 0.3 59.3 ± 1.7 71.5 ± 0.5
Sedum n-Hex – H -2.9 ± 1.3 13.8 ± 1.0 51.1 ± 2.0 6.7 ± 1.5 8.2 ± 0.6 39.2 ± 1.8 dendroideum (10) EtOH – E -0.3 ± 1.6 8.4 ± 0.6 16.9 ± 1.1 5.6 ± 1.1 4.9 ± 0.8 8.6 ± 0.4
Waltheria n-Hex – H -16.1 ± 0.8 11.6 ± 1.1 70.4 ± 1.8 6.6 ± 0.8 15.0 ± 1.2 45.5 ± 1.0 douradinha (11 ) EtOH – E 28.6 ± 0.9 79.9 ± 1.5 84.1 ± 0.4 33.9 ± 1.3 76.3 ± 0.8 80.2 ± 1.4
Xanthium n-Hex – H 12.3 ± 1.1 64.5 ± 2.2 83.6 ± 1.8 55.4 ± 0.6 55.2 ± 0.7 48.1 ± 1.6 cavanillesii (12) EtOH – E 10.0 ± 1.5 85.7 ± 0.2 81.4 ± 1.8 13.1 ± 1.3 42.9 ± 1.7 63.4 ± 1.7
The results were expressed in percentage and represent the mean ± S.E.M. of two independent experiments. For release of elastase resveratrol was used as a standard at concentrations of 10 and 20 µM, the average was 80.59 ± 0.86 and 87.28 ± 0.64, and for direct inhibition GW311616A at a concentration of 20, 50 and 100 nM, the data were 35.20 ± 2.42, 50.55 ± 1.66 and 68.47 ± 0.62, respectively
34 Results
2.3. Peltodon longipes
During the screening phase the n-hexane extract of Peltodon longipes showed a high cytotoxicity, caspase-3 activation and antibacterial activity. Therefore, the plant was selected for further extensive phytochemical and biological studies.
2.3.1. Isolation procedure
The n-hexane extract prepared from the roots of P. longipes was investigated by LC-NMR analysis as described in section 5.2.1. and six known diterpenes from the abietane type were identified as royleanone (I), 7 α-acetoxyroyleanone (II), 6 α-hydroxyroyleanone (III), 7 α and β- hydroxyroyleanone (IV and V) and inuroyleanol (VI). They were assigned in the HPLC chromatogram to the respective peaks as shown in Figure 2.7.
IV
VI II
V
I III
Figure 2.7 - HPLC chromatogram of the n-hexane extract of Peltodon longipes with the respective identified diterpene by LC-NMR analysis.
As these compounds are known for their interesting cytotoxic activity (Araujo et al. 2006; Areche et al. 2009; Jonathan et al. 1989; Kupchan et al. 1968; Slamenova et al. 2004), the n- hexane extract from the roots of Peltodon longipes was subjected to comprehensive Results 35 separation procedures. Fractionation with open column chromatography and low pressure liquid chromatography on silica gel was performed as described in section 5.2.2. The fractionation was monitored by TLC and HPLC. Twelve known diterpenes from the abietane type were isolated and identified based on mass spectra, 1D ( 1H and 13 C) and 2D NMR (COSY, HSQC and HMBC) analysis and comparison of their spectral data from the literature. Detailed information is given in the following chapters.
2.3.2. Structure elucidation of the isolated abietane diterpenes
2.3.2.1 7 ααα-acetoxyroyleanone (syn. 7-O-acetylhorminone)(1)
Figure 2.8 - Chemical structure of 7 α-acetoxyroyleanone.
Compound 1 (686.8 mg) (Figure 2.8) was isolated as described in section 5.2.2 and obtained as yellow amorphous solid. The EI-MS spectrum (Figure 2.9) displayed a molecular + + ion peak at m/z 374 [M] and further fragment ions at m/z =332 [M-COCH 2] and 314 [M- + H2O] which suggested the molecular formula C 22 H30 O5.
13 The C-NMR spectrum (Figure 2.10) displayed 22 carbons from which two ( δC 21.0 q and 1 δC 169.4 s) could be assigned to an acetyl group. Accordingly, in the H-NMR spectrum a three-proton singlet at δH 2.01 appeared (Figure 2.11). The remaining 20 signals indicated the presence of a diterpene. Three singlets for methyl groups at δH 0.90 (H-18), δH 0.89 (H-19) 36 Results
and δH 1.26 (H-20) as well as signals for an isopropyl-group at δ 3.17 (H-15), δ 1.19 (H-16), and δ 1.23 (H-17) suggested the presence of a diterpene from the abietane type. The HMBC spectrum (Figure 2.12) showed correlations between H-16 and H-17 with the signal at δC 124.6 which was assigned to C-13. Furthermore, long-range correlations were observed between H-15 and C-12 ( δC 150.7), C-13 ( δC 124.6) as well as C-14 ( δC 185.4), respectively. The chemical shift of δC 185.4 indicated the occurrence of a carbonyl group and that one of δC 150.7 of an oxygen function. C-12 was substituted with a hydroxy group, because of the singlet at δH 7.15, which disappeared by addition of D2O. The position of the hydroxyl group was confirmed by long-range correlations between the singlet at δH 7.15 and
C-11 ( δC 183.7), C-12 ( δC 150.7) and C-13 ( δC 124.6), respectively. A further signal in the low field occurred at δC 183.7, characteristic for a carbonyl group, and exhibited long-range correlations with the septet of the isopropyl group ( δH 3.17). Together with two further signals in the low field region at δC 139.4 (C-8) and at δC 149.9 (C-9) the presence of a tetra- substituted para -quinone system could be concluded.
19.8 OH 1.23 d
24.1 150.7 3.17qq 19.6 O 183.7 1.19 d 124.6
185.4
O
The observed correlations in the HMBC spectrum for H-20 ( δH 1.26 s) with C-1 ( δ 35.7 t),
C-5 ( δ 46.1 d), C-9 ( δ 149.9 s), and C-10 ( δ 39.0 s), for H-18 ( δH 0.90 s) with C-3 ( δ 40.9 t),
C-4 ( δ 32.9 s), C-5 ( δ 46.1 d), and C-19 ( δ 21.6 q) as well as for H-19 ( δH 0.89 s) with C-3, C- 4, C-5, and C-18 ( δ 32.9 q) enabled the assignment of the signals for the methyl groups and are in agreement with the proposed abietane-type diterpene. From the H,H-COSY (Figure 2.13), HSQC (Figure 2.14), and HMBC spectra and the observed coupling constants the signals for the methylene protons at C-2 ( δ 18.7), C-6 ( δ 24.6) and the secondary alcohol at C-7 ( δ 64.4) could be assigned (Table 2.3). The low field shifted signal for H-7 ( δΗ 5.95, dd) suggested that the hydroxyl was esterified. Accordingly, a long-range correlation could be observed between the CH 3-group of the acetyl moiety and C- Results 37
7 ( δC 64.4). From the coupling constants for H-7 ( J = 3.9 and 1.7 Hz) an α configuration of the acetyl group was deduced.
The assignment ( α and β) for the hydrogens at C-1, C-2, C-3, and C-6 was determined as follows: the coupling constants of H-5 ( J = 13.0 Hz and 1.2 Hz) are in agreement with a diaxial and an axial/equatorial coupling and suggested an axial position, H-6 at δΗ 1.63 (ddd, J = 14.7 Hz, 13.0 Hz and 3.9 Hz) must be β and axial , in accordance with the dihedral angles and the coupling constants and H-6 α corresponded to the signal at δΗ 1.96 (brd, J = 14.7 Hz). The assignment of the protons at C-1, 2 and 3 followed from the coupling constants. Assignments of the respective carbon atoms were confirmed by the HSQC-correlations.
H CH3 H CH3 H
H H 1 9 3 5 7 H
H H3C H H H OAc
Altogether, the 1D and 2D 1H- and 13 C-NMR data were in agreement with the occurrence of 7 α-acetoxyroyleanone and confirmed by the NMR data reported for this diterpene (Tezuka et al. 1998; Razak et al. 2010). This diterpene has been isolated from several Lamiaceae species. 38 Results
D:\icis\m fpbc92b2 07/24/08 09:57:37 AM PL-1 EI-direkt s66, Source:150',70eV,500uA ,m /z 41-800
m fpbc92b2 #1 RT: 2.43 AV: 1 NL: 2.38E6 T: + c EI Full m s [ 41.00-800.03] 314.1
2300000
2200000 332.1 2100000
2000000
1900000
1800000
1700000
1600000
1500000
1400000
1300000
1200000 Intensity 1100000
1000000 299.1 900000
800000 43.1
700000
600000 245.1
500000 244.1 333.2 400000 55.0 231.1 83.0 271.1 69.0 374.2 300000 217.1 258.1 91.0 108.9 186.9 200000 123.1 281.1 133.0 159.1 100000 334.2 375.2
0 50 100 150 200 250 300 350 400 450 500 m /z
Figure 2.9 - EI-MS spectrum of 7 α-acetoxyroyleanone (1). Results 39
13 Figure 2.10 - C-NMR spectrum of 7 α-acetoxyroyleanone (1) (100 MHz, CDCl 3).
40 Results
1 Figure 2.11 - H-NMR spectrum of 7 α-acetoxyroyleanone (1) (400 MHz, CDCl 3).
Results 41 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 18 19 1.0 17 16 20 3 1 3 5 1.5 2 6 2 6 2´ 2.0 2.5 1 3.0 15 3.5 4.0 ppm 4.5 5.0 5.5 7 6.0 6.5 7.0 12 OH at C 7.5 14 1´ 8 9 11 13 12 1 10 3 6/15 7 5 2/20 2´ 4/18 16/17 19
Figure 2.12 - HMBC spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3).
42 Results ppm
Figure 2.13 - H;H-COSY spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3). Results 43 15 20 25 30 35 40 45 50 55 60 65 70 0.5 18 19 1.0 17 16 20 3 1 3 5 1.5 2 6 2 2´ 6 2.0 2.5 1 3.0 15 ppm 3.5 4.0 4.5 5.0 7 5.5 12 OH at C 6.0 2´ 10 15 1 6 3 7 20 16/17 19 5 2 4/18
Figure 2.14 - HSQC spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3). 44 Results
Table 2.3 - NMR data of 7 α-acetoxyroyleanone (1) compared to the NMR data from the literature*. 1 Lit * 1 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] 2, 3, 10, 9, 5, α 1.25 m 1.22 td (13.5, 3.5) 20 1 35.7 t 35.8 2.73 dtd (13.5, 3.5, β 2.75 ddd (13.1, 3.4, 1) 1.5) α 1.60 m 1.57 dtd (13.5, 3.5) 2 18.7 t 18.8 1.75 ddddd (13.5, β 1.75 qt (13.5, 3.5) 13.5, 13.3, 3.6, 3.6) α 1.25 m 1.23 td (13.5, 3.5) 2, 1, 19, 5 3 40.9 t 41.0 1.49 dtd (13.5, 3.5, β 1.50 m 4, 18, 2, 19 1.5) 4 32.9 s 33.0 4, 18, 19, 10, 5 α 46.1 d 46.1 1.50 dd (13, 1.2) 1.48 dd (13, 1.5) 20, 6, 7 1.63 ddd (14.7, 13, β 1.61 ddd (14.5, 13, 4) 6 24.6 t 24.6 3.9) α 1.96 brd (14.7) 1.95 dt (14.5 1.5) 8, 7, 10 7 β 64.4 d 64.5 5.95 dd (1.7, 3.9) 5.94 dd (4, 1.5) 8 139.4 s 139.4 9 149.9 s 150.0 10 39.0 s 39.1 11 183.7 s 184.2 12 150.7 s 150.6 13 124.6 s 124.7 14 185.4 s 185.4 16, 17, 13, 15 24.1 d 24.1 3.17 septet (7) 3.16 heptet (7) 12, 14 16 19.6 q 19.7 1.19 d (7) 1.19 d (7) 17, 15, 13 17 19.8 q 19.9 1.23 d (7) 1.23 d (7) 15, 16, 13 18 32.9 q 33.0 0.90 s 0.89 s 3, 4, 5, 19 19 21.6 q 21.6 0.89 s 0.88 s 3, 4, 5, 18 20 18.4 q 18.5 1.26 s 1.24 s 10, 1, 5, 9
1´ 169.4 s 169.4
2´ 21.0 q 21.1 2.01 s 2.03 s 1´ OH at 7.15 s 7.18 brs 11, 12, 13 C12 * (Tezuka et al. 1998); ** HMBC correlations are from proton (s) stated to the indicated carbon.
Results 45
2.3.2.2 7 ααα-hydroxyroyleanone (syn horminone) (2)
Figure 2.15 - Chemical structure of 7 ααα-hydroxyroyleanone (2).
Compound 2 (65.5 mg) (Figure 2.15) was isolated as described in section 5.2.2 and obtained as yellow amorphous solid. The EI mass spectrum (Figure 2.16) displayed a + + molecular ion peak at m/z 332 [M] and further fragment ions at m/z 314 [M-H20] and m/z + 299 [M-H20-CH 3] and is in agreement with the molecular formula C 20 H28 O4. The structure of compound 2 was further elucidated by interpretation of 1D and 2D NMR data and by comparison of the spectral data from that of compound 1 and the literature (Tezuka et al. 1998). The NMR data of compound 2 (Figure 2.17 to Figure 2.21 and Table 2.4) were quite similar to that ones of compound 1 except for the absence of the acetyl-group and an upfield- shift of the signal for H-7 to δΗ 4.75 (dd, J = 4.5 Hz and 1.2Hz) indicating the presence of a free hydroxyl group. Thus, compound 2 was identified as 7 α-hydroxyroyleanone, which has already been found in Rodriguez 2003; Tezuka et al. 1998.
46 Results
D:\icis\m fpbd40b 02/03/09 09:39:44 AM PL-2 EI-direkt s28 , Source:200',70eV,500uA ,m/z 41-600
m fpbd40b #1 RT: 1.13 AV: 1 NL: 1.13E7 T: + c EI Full ms [ 41.00-600.01] 195.1
11000000
10500000
10000000 332.3 9500000
9000000
8500000
8000000
7500000
7000000 314.3 6500000
6000000 299.2 5500000 Intensity
5000000
4500000 261.2
4000000
3500000
3000000 317.2 277.2
2500000 123.2 210.2 245.2 333.3 2000000 219.2 235.2 179.1 1500000 289.2
1000000 152.1 161.1 69.2 83.2 91.1 109.2 135.1 500000 55.2 334.3 347.3 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m /z
Figure 2.16 - EI-MS spectrum of 7 ααα-hydroxyroyleanone (2).
Results 47
13 Figure 2.17 - C-NMR spectrum of 7 ααα-hydroxyroyleanone (2) (100 MHz, CDCl 3).
48 Results 0.5 1.0 1.5 19 1.0 18 6 2.0 2.5 1 1.20 1 16 3.0 20 15 17 1.25 3.5 3 4.0 ppm 3 1.5 5 2 1.6 4.5 6 7 1.7 5.0 2 1.8 5.5 6.0 17 16 O 15 14 13 6.5 OH 8 12 7 OH 6 11 9 7.0 10 5 O 20 12 C 18 OHat 1 4 19 2 3 7.5
1 Figure 2.18 - H-NMR spectrum of 7 ααα-hydroxyroyleanone (2) (400 MHz, CDCl 3).
Results 49 ppm
Figure 2.19 - HSQC spectrum of 7 ααα-hydroxyroyleanone (2) (CDCl 3).
50 Results ppm 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 19 1.0 18 1 3 17 20 16 3 5 1.5 2 6 2 6 2.0 2.5 1 3.0 15 3.5 4.0 ppm 4.5 7 5.0 5.5 6.0 6.5 7.0 12 7.5 9 8 11 14 13 12 10 1 15 3 5 20 7 18/4 2 6 19 16/17
Figure 2.20 - HMBC spectrum of 7 ααα-hydroxyroyleanone (2) (CDCl 3).
Results 51 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 0.8 19 18 1.0 1 1.2 3 17 20 16 1.4 3 5 2 1.6 6 2 1.8 6 2.0 2.2 2.4 2.6 1 2.8 3.0 ppm 15 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 7 4.8 5.0 5.2 5.4 3 2 3 1 5 1 6 6 15 7 2 17/20/16 19 18
Figure 2.21 - H;H-COSY spectrum of 7 ααα-hydroxyroyleanone (2) (CDCl 3).
52 Results
Table 2.4 - NMR data of 7 ααα-hydroxyroyleanone (2) compared to the NMR from the literature*. 2 Lit * 2 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.20 m 1.18 td (13.5, 3.5) 1 35.7 t 35.8 2.71 ddd (12.8, 3.1, β 2.70 dtd (13.5, 3.5, 1.5) 2.5) α 1.55 m 1.47 dtd (13.5, 3.5, 1.5) 4, 10 2 18.8 t 18.9 1.75 ddddd (13.7, β 1.73 qt (13.5, 3.5) 13.7, 13.7, 3.7, 3.7) α 1.27 m 1.25 td (13.5, 3.5) 2, 1, 4, 18 3 41.0 t 41.1 β 1.49 m (13.7, 12.8, 2) 1.56 dtd (13.5, 3.5) 4 33.0 s 33.0 4, 18, 19, 5 α 45.7 d 45.7 1.54 brd (13.7) 1.54 brd (13.5) 10, 20, 6, 7, 9 1.61 ddd (13.7, 13.7, β 1.61 ddd (13.5, 4.5) 5, 10 6 25.7 t 25.8 4.5) α 1.98 brd (13.7) 1.97 brd (14.5) 10, 5, 7, 8 5, 6, 8, 9, 7 β 63.1 d 63.2 4.75 dd (4.5, 1.2) 4.73 brd (4.5) 14 8 143.1 s 143.2 9 147.8 s 147.8 10 39.1 s 39.1 11 183.8 s 183.9 12 151.0 s 151.1 13 124.1 s 124.2 14 189.1 s 189.1 16, 17, 13, 15 23.9 d 24.0 3.18 septet (7) 3.16 heptet (7) 12, 14 16 19.7 q 19.8 1.22 d (7) 1.21 d (7) 17, 15, 13 17 19.8 q 19.9 1.24 d (7) 1.22 d (7) 16, 15, 13 18 33.1 q 33.1 1.0 s 0.98 s 19, 4, 3, 5 19 21.6 q 21.7 0.92 s 0.90 s 18, 4, 3, 5 20 18.3 q 18.4 1.23 s 1.21 s 10, 1, 5, 9
OH at C 7 3.07 brs OH at 11, 12, 13 7.26 s 7.33 brs C12 * (Tezuka et al. 1998); **HMBC correlations are from proton (s) stated to the indicated carbon.
Results 53
2.3.2.3 Royleanone (3)
Figure 2.22 - Chemical structure of royleanone (3).
Compound 3 (42.8 mg) (Figure 2.22) was isolated as described in section 5.2.2. and obtained as orange amorphous solid. The MS spectrum (Figure 2.23) gave a fragment ion + + peak at m/z 316 [M] and a fragment ion at m/z 299 [M- CH 3] which is in agreement with the 13 1 molecular formula C 20 H28 O3. Its C-NMR (Figure 2.24) and H-NMR spectra (Figure 2.25) were similar with that of compounds 1 and 2, but significant differences occurred for the chemical shifts of C-5, 6, 7, and 8 and the respective protons. Thus, signals for C-5 and C-8 were downfield-shifted, whereas those ones for C-6 and 7 upfield-shifted. Especially the signal for C-7 appeared in the low field ( δC 26.1) indicating that it was not substituted with a hydroxyl group. The two protons could be assigned for H-7β at δΗ 2.71 m and for H-7α at δΗ 2.35 (Figure 2.26, Figure 2.27 and Figure 2.28 and Table 2.5). All 1D and 2D NMR and MS data as well as literature data were in agreement with the occurrence of royleanone (Rodriguez 2003; Tezuka et al. 1998).
54 Results
D:\icis\m fpbd45b 03/03/09 10:32:30 AM PL-7 EI-direkt s23 , Source:200',70eV,500uA ,m/z 41-600 m fpbd45b #1 RT: 0.95 AV: 1 NL: 1.16E7 T: + c EI Full ms [ 41.00-600.01] 316.2 11500000
11000000
10500000
10000000
9500000
9000000
8500000
8000000
7500000
7000000
6500000
6000000
Intensity 5500000
5000000
4500000
4000000
3500000
3000000
2500000 205.1 317.3 314.2 2000000 301.2 245.2 1500000
1000000 244.2 330.3 187.1 283.2 298.2 213.1 259.2 185.1 500000 55.1 83.1 91.1 115.1 128.1 165.1 331.3 346.2 0 50 100 150 200 250 300 350 400 m /z
Figure 2.23 - EI-MS spectrum of royleanone (3).
Results 55
13 Figure 2.24 - C-NMR spectrum of royleanone (3) (100 MHz, CDCl 3). 56 Results
1 Figure 2.25 - H-NMR spectrum of royleanone (3) (400 MHz, CDCl 3). Results 57 10 15 20 25 30 35 40 45 50 55 19 0.9 18 5 1.1 1 3 17 16 20 1.3 6 3 1.5 2 1.7 2 6 1.9 2.1 ppm 2.3 7 2.5 2.7 7 1 2.9 3.1 15 3.3 3.5 10 6 15 3 2 7 1 5 19 4/18 16/17/20
Figure 2.26 - HSQC spectrum of royleanone (3) (CDCl3).
58 Results ppm
Figure 2.27 - HMBC spectrum of royleanone (3) (CDCl3). Results 59
Figure 2.28 - H;H-COSY spectrum of royleanone (3) (CDCl 3).
60 Results
Table 2.5 - NMR data of royleanone (3) compared to the NMR from the literature*. 3 Lit * 3 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.13 m 1.12 td (13.5, 3.5) 2, 10, 20, 9 1 36.2 t 36.2 2.78 ddd (13.5, 3.5, β 2.75 dtd (13.5, 3.5, 1.5) 5, 9 1.0) α 1.55 m 1.53 dq (13.5, 3.5) 2 18.8 t 18.9 β 1.73 m 1.72 qt (13.5, 3.5) α 1.21 m 1.20 m 2, 1, 4, 18, 19 3 41.2 t 41.3 β 1.48 m 1.46 dtd (13.5, 3.5, 1.5) 4 33.4 s 33.4 4, 18, 19, 10, 5 α 51.6 d 51.7 1.10 brd (13) 1.10 dd (12.5, 1.5) 20, 6, 7 1.38 dddd (13.5, 12.5, β 1.37 m 4, 10 6 17.4 t 17.4 11.5, 6) α 1.88 m 1.87 ddt (13.5, 7.5, 1.5) 8, 7, 5, 10 2.35 ddd (11.5, 7.2, α 2.34 ddd (21, 11.5, 7.5) 6, 8, 9 7 26.6 t 26.7 4.2) β 2.71 m 2.71 ddd (21, 6, 1.5) 6, 5, 8, 9 8 145.9 s 146.0 9 146.6 s 146.5 10 38.4 s 38.4 11 183.3 s 183.4 12 150.5 s 150.5 13 123.6 s 123.7 14 187.5 s 187.5 16, 17, 13, 15 24.0 d 24.1 3.17 septet (7) 3.15 heptet (7) 12, 14 16 19.8 q 19.9 1.23 d (7) 1.20 d (7) 13, 17, 15 17 19.9 q 20.0 1.24 d (7) 1.21 d (7) 15, 16, 13 18 33.4 q 33.5 0.95 s 0.93 s 19, 4, 3, 5 19 21.7 q 21.8 0.91 s 0.90 s 4, 18, 3, 5 20 20.0 q 20.0 1.27 s 1.25 s 10, 9, 1, 5 OH at 13, 12, 11 7.25 s 7.23 s C12 * (Tezuka et al. 1998); **HMBC correlations are from proton (s) stated to the indicated carbon.
Results 61
2.3.2.4 7-ketoroyleanone (syn 7-oxoroyleanon) (4)
Figure 2.29 - Chemical structure of 7-ketoroyleanone (4).
Compound 4 (10.5 mg) was isolated as described in section 5.2.2. and obtained as brown amorphous solid. Its chemical structure (Figure 2.29) was elucidated based on 1D and 2D NMR and MS spectral data. Moreover, NMR data were compared with those from compound 1 and 2 as well as from the literature (Rüedi 1984). The EI-MS spectrum (Figure 2.30) showed a molecular ion peak at m/z 330 [M] + and a fragment ion resulting from the loss of a methyl group at m/z 315 and was consistent with the molecular formula C 22 H26 O4. The presence of a p-quinone abietane system in 4 could be elucidated as done for compound 1. The molecular weight suggested the occurrence of a further oxygen function which must be a carbonyl group because of the signal at δC 196.8 (Figure 2.31 and Figure 2.32). Long-range correlations between δC 196.8 and the signals for 6 α and 6 β as well as downfield-shifts of the signals for C-6 and C-9 and an upfield-shift of C-8 compared to the respective signals of compound 3 were in agreement with a carbonyl group at C-7 (see Figure 2.33 and Figure 2.34). Consequently, protons at C-6 showed at geminal coupling and a vicinal coupling with H-5 (H-6α: δ 2.73 dd, J = 17.9 Hz and 3.3; H-6β: δ 2.55 dd, J = 17.9 Hz and 14.4) (Table 2.6). All spectral data (1 and 2D NMR and MS) agreed with the occurrence of 7- ketoroyleanone (Rüedi 1984) that was identified for the first time in Bhat et al. 1975. 62 Results
D:\icis\m fpbd59b 04/02/09 10:39:10 AM PL-10 EI-direkt s48 , Source:200',70eV,500uA ,m/z 41-800 m fpbd59b #1 RT: 1.79 AV: 1 NL: 1.02E7 T: + c EI Full ms [ 41.00-799.99] 330.2
10000000
9500000
9000000 315.1 8500000
8000000
7500000
7000000
6500000
6000000
5500000
5000000 Intensity
4500000
4000000
3500000
3000000 245.1
332.2 2500000 247.2
2000000 233.1 259.2 1500000 273.2 1000000 219.1 287.2 297.2 91.1 149.1 203.1 83.1 109.1 193.1 333.3 500000 55.1 123.1 175.1 95.1 347.3 0 50 100 150 200 250 300 350 400 450 m /z
Figure 2.30 - EI-MS spectrum of 7-ketoroyleanone (4).
Results 63
13 Figure 2.31 - C-NMR spectrum of 7-ketoroyleanone (4) (100 MHz, CDCl 3). 64 Results 0.5 1.0 1.5 3 2 2 5 0.9 2.0 18 1.0 19 2.5 6 1.2 6 1 16 3.0 17 3 1.3 1 15 20 1.4 3.5 4.0 ppm 4.5 5.0 5.5 6.0 17 16 O 15 6.5 14 13 O 8 12 7 OH 12 7.0 6 11 C 9 OHat 10 5 O 20 18 1 4 7.5 19 2 3
1 Figure 2.32 - H-NMR spectrum of 7-ketoroyleanone (4) (400 MHz, CDCl 3).
Results 65 15 20 25 30 35 40 45 50 55 18 19 1.0 3 17 1.2 16 1 20 1.4 3 1.6 2 2 1.8 5 2.0 ppm 2.2 2.4 6 2.6 6 2.8 1 3.0 3.2 15 3.4 10 4 6 5 3 2 19 20 1 18 15 16/17
Figure 2.33 - HSQC spectrum of 7-ketoroyleanone (4) (CDCl 3).
66 Results 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 18 1.0 19 1 3 16 17 20 1.5 3 2 2 5 2.0 2.5 6 6 1 3.0 15 3.5 4.0 ppm 4.5 5.0 5.5 6.0 6.5 12 C OH at OH 7.0 7.5 8 7 13 9 11 14 12 10 4 6 20 5 19 3 18 1 2 15 16/17
Figure 2.34 - HMBC spectrum of 7-ketoroyleanone (4) (CDCl 3).
Results 67
Table 2.6 - NMR data of 7-ketoroyleanone (4) compared to the NMR from the literature*. 4 Lit * 4 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.13 m 1 35.5 t 35.1 β 2.90 brd (13.5) 2.86 brd (13) α 1.65 m 2 18.2 t 18.4 β 1.80 m α 1.29 m 2, 1, 19 3 40.7 t 40.7 β 1.55 brd (13.0) 2 4 33.2 s 33.1 20, 19, 4, 18, 1, 5 α 48.9 d 48.9 1.84 dd (14.3, 3.3) 1.81 dd (18.6) 6, 10, 3, 9, 7 β 2.55 dd (17.9, 14.4) 2.50 dd (18.3, 14) 7, 5, 10 6 36.7 t 36.3 α 2.73 dd (17.9, 3.3) 2.67 dd (18.3, 4.6) 7, 5, 10 7 196.8 s 196.5 8 133.0 s 132.9 9 155.3 s 155.2 10 39.9 s 39.8 11*** 184.7 s 184.6 12 150.7 s 150.6 13 125.0 s 125.3 14*** 184.9 s 184.9 15 24.2 d 24.1 3.17 septet (7) 3.18 heptet (7) 16, 17, 13, 12, 14 16*** 19.7 q 19.6 1.23 d (7) 1.26 d (7) 13, 17, 15 17*** 19.7 q 19.6 1.24 d (7) 1.23 d (7) 15, 16, 13 18 32.6 q 32.5 0.95 s 0.92 s 19, 4, 3, 5 19 21.2 q 21.1 0.91 s 0.96 s 4, 18, 3, 5 20 17.9 q 17.8 1.27 s 1.35 s 10, 9, 1, 5 OH at 7.25 s 7.02 s 13, 12, 11 C12 *(Rüedi 1984); **HMBC correlations are from proton (s) stated to the indicated carbon; *** assignments interchangeable.
68 Results
2.3.2.5 7 ααα-ethoxyroyleanone (5)
Figure 2.35 - Chemical structure of 7 α-ethoxyroyleanone (5).
Compound 5 (12.1 mg) (Figure 2.35) isolated as described in section 5.2.2 was obtained as light orange amorphous solid. The EI-MS spectrum (Figure 2.36) presented a molecular ion + + + peak at m/z 360 [M] and further fragment ions at m/z 345 [M-CH 3] , 331 [M-CH 3CH 2] and + 316 [M-CH 3CH 2-CH 3] which was coherent with the molecular formula C 22 H32 O4. The presence of an ethoxy group was confirmed by signals at δC 65.4 and δC 15.7 and a multiplet at δH 3.71 and a triplet at δH 1.23. A long-range was observed between C-7 ( δC 69.2) and H-1’ and was in agreement with its location at C-7. 1D and 2D 1H- and 13 C-NMR were similar to that ones of compound 2 and agreed well with those reported in the literature for 7 α- ethoxyroyleanone (Michavila et al. 1986) (see Figure 2.37 to Figure 2.41 and Table 2.7). Results 69
D:\icis\mfpbd85b 05/27/09 12:12:08 PM PE-1 EI-direkt s47, Source:200',70eV,500uA ,m/z 41-600 m fpbd85b #1 RT: 1.75 AV: 1 NL: 7.33E6 T: + c EI Full ms [ 41.00-600.01] 316.1
7000000
6500000
6000000
5500000
5000000 331.1
4500000
4000000
Intensity 3500000 360.1
3000000
245.0 2500000
220.0
2000000 299.1 221.0 247.1 232.0 345.1 1500000
219.0 271.1 1000000 218.0 361.1 187.0
500000 109.0 173.0 82.9 159.0 69.0 90.9 145.0 55.0 362.2 0 50 100 150 200 250 300 350 400 450 m /z
Figure 2.36 - EI-MS spectrum of 7 α-ethoxyroyleanone (5).
70 Results
13 Figure 2.37 - C-NMR spectrum of 7 α-ethoxyroyleanone (5) (100 MHz, CDCl 3).
Results 71
1 Figure 2.38 - H-NMR spectrum of 7 α-ethoxyroyleanone (5) (400 MHz, CDCl 3).
72 Results
Figure 2.39 - HSQC spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3). Results 73
Figure 2.40 - HMBC spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3).
74 Results ppm
Figure 2.41 - H;H-COSY spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3).
Results 75
Table 2.7 - NMR data 7 α-ethoxyroyleanone (5) compared to the NMR from the literature*. 5 5 Lit * 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.25 m 2, 10, 20, 5, 9 1 35.5 t β 2.70 brd (12.4) 2.68 ddd (12, 4, 2) α 1.58 m 2 18.8 t β 1.75 m α 1.27 m 2, 1, 4, 19, 18, 5 3 40.8 t β 1.48 brd (13.0) 4 33.1 s 4, 18, 19, 10, 20, 5 α 45.4 d 1.65 dd (12.7, 1.2) 1.63 dd (12.9, 1.5) 9, 6, 7 β 1.38 m 1.37 ddd (3.5) 6 22.9 t α 2.05 brd (14.2) 2.05 ddd (14.2, 1.8) 7, 8, 5, 4, 10 7 β 69.2 d 4.40 dd (1.8, 1.6) 4.42 dd 5, 8, 9, 1` 8 141.6 s 9 147.8 s 10 39.9 s 11 184.1 s 12 150.6 s 13 124.6 s 14 186.4 s 15 24.2 d 3.20 m 3.17 septet (7) 16, 17, 13, 12, 14 16 19.9 q 1.22 d (7) 1.23 d (7) 13, 17, 15 17 19.7 q 1.20 d (7) 1.19 d (7) 13, 16, 15 18 32.9 q 0.97 s 0.94 s 19, 3, 4, 5 19 21.8 q 0.92 s 0.91 s 18, 4, 3, 5 20 18.5 q 1.23 s 1.21 s 1, 10, 5, 9
1` 65.4 t 3.71 m 3.71 dq , 3.68 dq (8.9, 7.1) 7, 2` 2` 15.7 q 1.23 t (7) 1.21 t 1` OH at 7.11 s 7.13 s 13, 12, 11 C12 * (Michavila et al. 1986); **HMBC correlations are from proton (s) stated to the indicated carbon.
76 Results
2.3.2.6 Iguestol (6)
O 16
12 15 HO 17 11 13
20
1 9 14
2 10 8
3 5 7 4 6
19 18 OH
Figure 2.42 - Chemical structure of iguestol (6).
Diterpene 6 (17.5 mg) (Figure 2.42) was isolated as described in section 5.2.2 and obtained as light yellow amorphous solid. In the EI-MS spectrum (Figure 2.43) a molecular ion peak + was present at m/z 332 [M] which suggested the molecular formula C 21 H32 O3. The 1H-NMR (Figure 2.44) and 13 C-NMR spectra (Figure 2.45) of compound 6 showed some similarities with that ones from compounds 1 to 5 (see Table 2.8), such as the presence of an isopropyl group and three angular methyl groups indicating an abietane-type diterpene. 13 However, the typical p-quinone-moiety was missing in C-NMR spectrum . A singlet at δH 6.50 suggested an aromatic C ring in the abietane skeleton and was assigned to C-14 due to the long-range correlations with C-7 ( δC 42.2), C-8 ( δC 131.2), C-9 ( δC 131.9), and C-12 ( δC 143.0). The aromatic ring was further substituted by a hydroxyl group which was located at
C-11 because of long-range correlations between δH 6.04 and C-8, C-9, C-11, and C-12. Additionally, a methoxy group at C-12 was observed in which the position was deduced from the long-range correlation with C-12. The long range correlations between the methyl group at δ 1 .17 (Me-18) as well as δ 1 .20 (Me-19) and C-4 ( δC 34.0) and C-5 ( δC 58.2) confirmed the correct assignments of C-4 and C-5. The signals of the corresponding protons were deduced from the HSQC spectrum (Figure 2.46). Long-range correlations between δΗ 1 .37 (Me-20) and C-1, C-4, C-5, C-8, and C-9 were used for the assignment of these carbon atoms. Results 77
The correlations in the HMQC (Figure 2.47) allowed the correct assignment of the corresponding protons.
From the H;H-COSY (Figure 2.48) the coupling system consisting of H-5 ( δΗ 1.44 d ( J =
9.2 Hz)), H-6 ( δΗ 4.26 ddd ( J = 9.2 Hz, 7.4 Hz and 5.5 Hz)) and H-7α ( δΗ 2.78 dd ( J = 15.8
Hz and 7.4 Hz)) and H-7β ( δΗ 3.18 dd ( J = 15.9 Hz and 5.5 Hz)) could be elucidated. The long-range correlations between H-7β and C-5 ( δC 58.2), C-6 ( δC 68.5), C-8 ( δC 131.2), C-9
(δC 131.9), and C-14 ( δC 117.4) confirmed the assignments. The stereochemistry of the secondary alcohol at C-6 was determined as α-equatorial on the basis of the coupling constant observed with H-5 ( δH 1.44, J = 9,2 Hz). Altogether 1D and 2D NMR data agreed well with those reported for 6 α,11-dihydroxy-12-methoxy-abieta-8,11,13-triene named as iguestol (Fraga et al. 2005).
D:\icis\m fpbd42b 02/18/09 08:43:29 AM PL-4 EI-direkt s37 , Source:200',70eV,500uA ,m/z 41-600 m fpbd42b #1 RT: 1.43 AV: 1 NL: 1.06E7 T: + c EI Full ms [ 41.00-600.01] 332.3 10500000
10000000
9500000
9000000
8500000
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7500000
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6500000 299.2 6000000
5500000 267.2 Intensity 5000000
4500000
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3500000
3000000
2500000 229.1 333.3
330.2 2000000 193.1 257.2 1500000 317.3 205.1 268.2 179.1 1000000 217.1 245.2 157.1 177.1 145.1 273.2 500000 69.1 95.1 115.0 334.3 55.1 289.2 0 50 100 150 200 250 300 350 400 450 m /z Figure 2.43 - EI-MS spectrum of iguestol (6).
78 Results
1 Figure 2.44 - H-NMR spectrum of iguestol (6) (400 MHz, CDCl 3).
Results 79
13 Figure 2.45 - C-NMR spectrum of iguestol (6) (100 MHz, CDCl 3).
80 Results ppm 10 15 20 25 30 35 40 45 50 55 60 65 70 0.8 19 1.0 18 1.2 3 17 16 20 1.4 5 3 1 2 1.6 2 1.8 2.0 2.2 2.4 2.6 ppm 7 2.8 3.0 1 7 3.2 15 3.4 3.6 12 C OCH3 at 3.8 4.0 4.2 6 4.4 10 4 12 C 1 OCH3 at 5 7 20 6 18 3 2 15 19 16/17
Figure 2.46 - HSQC spectrum of iguestol (6) (CDCl 3). Results 81 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 19 18 1.0 16 17 3 20 5 3 1.5 1 2 2 2.0 2.5 7 3.0 1 7 15 3.5 12 C OCH3 at ppm 4.0 6 4.5 5.0 5.5 11 C OH at OH 6.0 14 6.5 12 9 8 11 13 10 4 12 1 14 5 2 6 15 18 7 3 OCH3 at C 20 19 16/17
Figure 2.47 - HMBC spectrum of iguestol (6) (CDCl 3). 82 Results
Figure 2.48 - H;H-COSY spectrum of iguestol (6) (CDCl 3).
Results 83
Table 2.8 - NMR data iguestol (6) compared to the NMR data from the literature*. 6 Lit * 6 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.55 m 1.53 m 2, 3, 10 1 37.7 t 37.4 3.04 ddd (12.7, 2.6, β 2.99 dt (13, 4) 2, 10, 3 2.6) α 1.59 m 1.53 m 4, 10, 3 2 19.1 t 19.1 β 1.71 m 1.70 m - α 1.27 m 1.25 td (13, 3.3) - 3 42.6 t 42.6 β 1.49 m 1.45 dt (13, 4) - 4 34.0 s 34.0 20, 19, 4, 18, 1, 5 α 58.2 d 58.2 1.44 d (9.2) 1.39 d (9) 10, 7, 3, 6, 9 4.26 ddd (9.2, 7.4, 6 β 68.5 d 68.5 4.21 br (18) 8 5.5) α 2.78 dd (15.8, 7.4) 2.74 dd (15.7, 7.6) 5, 6, 8, 9, 14 7 42.2 t 42.2 β 3.18 dd (15.9, 5.5) 3.14 m 5, 6, 8, 9, 14 8 131.2 s 131.8 9 131.9 s 131.1 10 41.6 s 41.5 11 146.1 s 146.0 12 143.0 s 143.0 13 138.1 s 138.0 14 117.4 d 117.3 6.50 s 6.45 s 15, 7, 8, 9, 12 16, 17, 12, 13, 15 26.4 d 26.3 3.21 septet (7) 3.14 m 14 16 23.6 q 23.5 1.23 d (7) 1.18 d (7) 13, 15, 17 17 23.7 q 23.6 1.24 d (7) 1.20 d (7) 13, 15, 16 18 35.6 q 35.6 1.17 s 1.13 s 3, 4, 5 19 23.0 q 22.9 1.20 s 1.16 s 4, 18, 3, 5 20 21.3 q 21.2 1.37 s 1.33 s 1, 10, 5, 9 CH O 3 61.7 q 3.75 s 3.72 s 12 at C 12 OH at 6.04 s 5.98 s 9, 11, 12 C11
* (Fraga et al. 2005); **HMBC correlations are from proton (s) stated to the indicated carbon.
84 Results
2.3.2.7 Deoxyneocryptotanshinone (7)
Figure 2.49 - Chemical structure of deoxyneocryptotanshinone (7).
Compound 7 (6.2 mg) (Figure 2.49) was isolated as described in section 5.2.2 and obtained as brown to red amorphous solid. The EI-MS spectrum (Figure 2.50) showed a molecular ion + 13 peak at m/z 298 [M] that suggested the molecular formula C 19 H22 O3. The C-NMR spectrum (Figure 2.51) exhibited 19 signals, two of them were superimposed. One methyl group was missing compared to diterpenes 1 - 6. The presence of an isopropyl-group, a p-quinone structural element and an aromatic hydroxy group in the vicinity of the p-quinone system were obvious from the respective signals in the 13 C-NMR and 1H-NMR spectra (Figure 2.52 and Table 2.9). The two methyl groups were detected at δC 31.7 and δC 31.7 suggesting a nor- abietane diterpene type. They were bound to C-4, because of long-range correlation between
H-18/H-19 ( δH 1.34) and C-4 ( δC 34.7) as well as C-3 ( δC 37.7). Further assignments were based on long-range correlations (Figure 2.53) between H-1 ( δH 3.28) and C-2 ( δC 19.0), C-3
(δC 37.7), C-5 ( δC 152.4), C-9 ( δC 126.0), and C-10 ( δC 140.6), between H-6 ( δH 7.75) and C-
4 ( δC 34.7), C-8 ( δC 132.6), and C-10 ( δC 140.6) as well as between H-7 ( δH 8.02) and C-5 ( δC
152.4 s), C-9 ( δC 126.0), and C-14 ( δC 184.5). Chemical shift from C-5 - C-10 and from H-6 and H-7 were in a range typical for an aromatic ring. Signals from H-2 and H-3 were taken from the H;H-COSY (Figure 2.54) and HSQC spectra (Figure 2.55). Thus, compound 7 was unambiguously identified as deoxyneocryptotanshinone, which has already been found in Results 85
Salvia miltiorrhiza (Hayashi & Kakisava 1970; Ikeshiro et al. 1991). However, complete 1H- and 13 C-NMR data have not yet been reported.
D:\icis\m fpbd43b 03/03/09 10:28:00 AM PL-5 EI-direkt s17 , Source:200',70eV,500uA ,m/z 41-600
m fpbd43b #1 RT: 0.90 AV: 1 NL: 1.08E7 T: + c EI Full ms [ 41.00-600.01] 298.2
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2000000
1500000 229.1 1000000 255.2 165.1 201.1 213.1 265.2 128.1 254.1 314.2 500000 115.1 141.1 55.1 83.1 95.1 316.3 346.2 0 50 100 150 200 250 300 350 400 m /z
Figure 2.50 - EI-MS spectrum of deoxyneocryptotanshinone (7).
86 Results 10 2 20 16 17 15 1 30 18 19 4 3 40 50 60 70 3 CDCl 80 90 100 ppm 110 120 7 13 9 130 8 6 140 10 150 5 12 17 160 16 O 15 14 170 13 8 7 12 OH 180 6 11 11 9 14 5 10 O 18 190 4 1 19 2 3 200
13 Figure 2.51 - C-NMR spectrum of deoxyneocryptotanshinone (7) (100 MHz, CDCl 3). Results 87 0.0 1.2 0.5 16 1.3 17 18 19 1.0 1.4 1.5 3 2 2.0 2.5 3.0 1 15 3.5 4.0 ppm 4.5 5.0 5.5 6.0 6.5 17 16 O 15 7.0 14 13 8 7 12 OH 7.5 12 C 6 11 OHat 6 9 5 10 O 7 18 8.0 4 1 19 2 3 8.5
1 Figure 2.52 - H-NMR spectrum of deoxyneocryptotanshinone (7) (400 MHz, CDCl 3).
88 Results 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 0.5 17 1.0 16 18 19 1.5 3 3 2 2 2.0 2.5 3.0 1 1 15 3.5 4.0 ppm 4.5 5.0 5.5 6.0 6.5 7.0 7.5 12 C 6 OH at 7 8.0 14 8 5 11 9 10 12 4 6 3 1 15 2 7/13 18/19 16/17
Figure 2.53 - HMBC spectrum of deoxyneocryptotanshinone (7) (CDCl 3). Results 89
Figure 2.54 - H;H-COSY spectrum of deoxyneocryptotanshinone (7) (CDCl 3).
90 Results 20 30 40 50 60 70 80 90 100 110 120 130 17 1.0 16 18 19 1.5 3 3 2 2 2.0 2.5 3.0 1 1 15 3.5 4.0 ppm 4.5 5.0 5.5 6.0 6.5 7.0 7.5 12 C 6 OH at 7 8.0 8 9 4 6 1 3 2 15 7/13 18/19 16/17
Figure 2.55 - HSQC spectrum of deoxyneocryptotanshinone (7) (CDCl 3).
Results 91
Table 2.9 - NMR data of deoxyneocryptotanshinone (7). 7 7 1H δ/ppm, Position 13 C δ /ppm HMBC * multiplicity J [Hz] α 3.28 t (6.4) 2, 3, 9, 10, 5 1 29.8 t β 3.28 t (6.4) 2, 3, 9, 10, 5 α 1.85 m 4, 10, 1, 3 2 19.0 t β 1.85 m 4, 10, 1, 3 α 1.70 m 2, 1, 18, 19, 4, 5 3 37.7 t β 1.70 m 2, 1, 18, 19, 4, 5 4 34.7 s 5 152.4 s 6 133.3 d 7.75 d (8.2) 4, 8, 10 7 124.9 d 8.02 d (8.2) 9, 5, 14 8 132.6 s 9 126.0 s 10 140.6 s 11 183.3 s 12 153.1 s 13 124.9 s 14 184.5 s 15 24.3 d 3.38 septet (7) 16, 17, 13, 12, 14 16 19.8 q 1.30 d (7) 13, 17, 15 17 19.8 q 1.32 d (7) 16, 13, 15 18 31.7 q 1.34 s 5, 3, 4, 19 19 31.7 q 1.34 s 5, 3, 4, 18
OH at C 12 7.77 s 13, 12, 11 *HMBC correlations are from proton (s) stated to the indicated carbon.
92 Results
2.3.2.8 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8)
16 OH
12 15 O 11 17 20 13
1 9 14 2 10 8
3 4 5 7 6 O
19 18
Figure 2.56 - Chemical structure of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8).
Isolation of compound 8 (10.1 mg) (Figure 2.56) is described in section 5.2.2. A light yellow amorphous solid was obtained. The EI-MS spectrum (Figure 2.57) exhibited a similar pattern as the other isolated diterpenes with a molecular ion peak at m/z 330 [M] + and a + further intense fragment ion peak at m/z 315 [M-CH 3] suggesting the molecular formula
C21 H30 O3. 13 C-NMR (Figure 2.58) and 1H-NMR (Figure 2.59) data were similar to that ones of 6, but with an additional signal at δC 198.5 indicating the presence of a carbonyl group. A signal for a methine group was missing. A long-range correlation (Figure 2.60 and Figure 2.61) between
δC 198.5 and the protons at C-6 ( δH 2.68 and 2.58) as well as correlations between H-5 ( δH 1.88) and H-6 in the H;H-COSY spectrum (Figure 2.62) led to the conclusion that the carbonyl should be located at C-7. The 1H-NMR spectrum showed the signal for a methoxy group at δH 3.81. Long-range correlation between δH 3.81 and C-11 ( δC 144.09) confirmed its location at C-11. Accordingly, long-range correlations between δH 6.10 and C-11 ( δC 144.0),
C-12 ( δC 152.6), and C-13 ( δC 133.8) were in agreement with a hydroxyl group at C-12 (Table 2.10). Altogether, compound 8 was identified as 12-hydroxy-11-methoxyabieta-8,11,13-trien- 7-one, a diterpene which was already known from the Taxodiaceae Cryptomeria japonica . Its 1D and 2D 1H- and 13 C-NMR as well as MS data agreed well with the compound isolated in Results 93 this thesis (Su et al. 1996). The chemical shifts reported for C-11 and C-12 have to be exchanged.
D:\icis\m fpbd41b 02/18/09 08:40:19 AM PL-3 EI-direkt s49 , Source:200',70eV,500uA ,m/z 41-600 m fpbd41b #1 RT: 1.83 AV: 1 NL: 1.15E7 T: + c EI Full ms [ 41.00-600.01] 330.3 11500000
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Intensity 5500000
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4500000 245.1 4000000 193.1
3500000
3000000 331.3 2500000 247.2 233.1 2000000 273.2 299.2 1500000 219.0 203.1 267.2 1000000 191.1 283.2 333.3 128.1 141.0 177.1 500000 55.1 69.1 83.1 115.0 334.3 377.2 0 50 100 150 200 250 300 350 400 450 m /z
Figure 2.57 - EI-MS spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8).
94 Results
Figure 2.58 - 13 C-NMR spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (100 MHz, CDCl 3). Results 95
Figure 2.59 - 1H-NMR spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (400 MHz, CDCl 3). 96 Results 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 18 1.0 19 16 17 3 20 3 1.5 1 2 2 5 2.0 2.5 6 6 1 3.0 15 11 3.5 C OCH3 at 4.0 ppm 4.5 5.0 5.5 6.0 12 C OH at OH 6.5 7.0 7.5 14 9 11 8 7 12 13 10 4 2 14 6 3 1 15 5 11 C OCH3 at 18 20/19 17/16
Figure 2.60 - HMBC spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3). Results 97
Figure 2.61 - HSQC spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3). 98 Results 1 2 3 4 5 6 7 8 18 1.0 19 16 17 3 20 3 1.5 1 2 2 5 2.0 2.5 6 6 1 3.0 15 11 3.5 C OCH3 at 4.0 ppm 4.5 5.0 5.5 6.0 12 C OH at 6.5 7.0 7.5 14 8.0 1 2 3 15 5 2 6 6 1 12 C 7 OH OH at 11 17/16 OCH3 at C 3 18 19 20
Figure 2.62 - H;H-COSY spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3). Results 99
Table 2.10 - NMR data of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) compared to the NMR from the literature*. 8 Lit * 8 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.62 m 2 1 37.2 t 37.3 2.97 ddd (13.7, 3.7, β 2.92 brd (13.5) 3.1) α 1.67 m 2 19.1 t 19.1 1.77 ddddd (13.7, β 13.7, 13.7, 3.7, 3.7) α 1.28 m 19 3 41.0 t 41.0 β 1.52 m 4 33.5 s 33.6 4, 3, 18, 19, 10, 5 α 49.9 d 49.9 1.88 dd (13.8, 3.9) 1.83 dd (13.5, 4.5) 20, 9, 6, 7 α 2.68 dd (17.9, 3.9) 2.64 dd (18, 4.5) 7, 5, 10 6 35.5 t 35.6 β 2.58 dd (17.8, 13.8) 2.53 dd (18, 13.5) 7, 5, 10 7 198.5 s 198.7 8 124.9 s 124.9 9 145.4 s 144.0 10 40.2 s 40.2 11 144.0 s 152.6 12 152.6 s 145.4 13 133.8 s 133.9 14 122.8 d 122.8 7.85 s 7.79 s 15, 7, 9, 12 15 27.2 d 27.2 3.25 septet (7) 3.20 sept (7, 7) 16, 17, 12, 13, 14 16 22.3 q 22.1 1.25 d (7) 1.21 d (7) 15, 13, 17 17 22.1 q 22.3 1.28 d (7) 1.24 d (7) 15, 13, 16 18 33.0 q 33.0 0.95 s 0.91 s 19, 4, 3, 5 19 21.6 q 21.6 1.02 s 0.97 s 18, 4, 3, 5 20 21.2 q 21.2 1.40 s 1.36 s 10, 1, 5, 9 CH O at 3 61.4 q 61.4 3.81 s 3.76 s 11 C11 OH at 6.10 s 6.06 s 11, 12, 13 C12
* (Su et al. 1996); **HMBC correlations are from proton (s) stated to the indicated carbon.
100 Results
2.3.2.9 Inuroyleanol (9)
Figure 2.63 - Chemical structure of inuroyleanol (9).
Diterpene 9 (11.2 mg) (Figure 2.63) was isolated as described in section 5.2.2 and obtained as yellow to brown amorphous solid. The EI-MS spectrum (Figure 2.64) displayed a + molecular ion peak at m/z 346 [M] that corroborated with the molecular formula C 21 H30 O4. The 13 C-NMR (Figure 2.65) and 1H-NMR spectra (Figure 2.66) were similar to that one of compound 8. Pronounced differences in the chemical shifts occur for C-14 ( δC 158.2), and to a lesser degree for C-8 and C-9. Moreover, instead of a singlet for an aromatic proton a signal for a phenolic hydroxy group appeared at δH 13.40 which exhibited long-range correlations with C-8 ( δC 112.4), C-12 ( δC 152.1), C-13 ( δC 126.1), and C-14 ( δC 158.2). Therefore, this additional hydroxyl group should be located at C-14. Long-range correlations between the singlet for C-11-OH ( δH 5.70) and C-9, C-11 and C-12 as well as between the three proton singlet for the methoxy group at δH 3.81 and C-12 suggested the position of the methoxy group at C-12 (see Figure 2.67, Figure 2.68, Figure 2.69 and Table 2.11). Moreover, substitution of C-7 with a carbonyl group was confirmed by a long-range correlation between H-6 α/H-β and C-7. Hence, based on 1D and 2D NMR and MS data compound 9 was identified as inuroyleanol, which was also in agreement with the NMR data reported for this diterpene (Fraga et al. 2005). Inuroyleanol has already been isolated from Salvia broussonetii . Results 101
D:\icis\m fpbd44b 03/03/09 10:30:13 AM PL-6 EI-direkt s25 , Source:200',70eV,500uA ,m/z 41-600 m fpbd44b #1 RT: 1.02 AV: 1 NL: 1.09E7 T: + c EI Full ms [ 41.00-600.01] 346.2
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1500000 261.2 263.2 1000000 249.1 207.1 298.2 175.1 201.1 289.2 500000 299.2 348.3 215.1 247.1 55.1 69.1 83.1 115.1 128.1 173.1 0 50 100 150 200 250 300 350 400 450 m /z
Figure 2.64 - EI-MS spectrum of inuroyleanol (9).
102 Results
13 Figure 2.65 - C-NMR spectrum of inuroyleanol (9) (100 MHz, CDCl 3). Results 103
1 Figure 2.66 - H-NMR spectrum of inuroyleanol (9) (400 MHz, CDCl 3).
104 Results ppm
Figure 2.67 - HMBC spectrum of inuroyleanol (9) (CDCl 3). Results 105 10 15 20 25 30 35 40 45 50 55 60 65 18 1.0 19 1.2 3 20 1 1.4 17 16 3 1.6 2 2 1.8 5 2.0 2.2 ppm 2.4 6 2.6 6 2.8 3.0 3.2 1 15 3.4 3.6 12 C OCH3 at 3.8 10 4 1 3 5 18 15 6 2 12 20 C 19 OCH3 at 16/17
Figure 2.68 - HSQC spectrum of inuroyleanol (9) (CDCl 3). 106 Results
Figure 2.69 - H;H-COSY spectrum of inuroyleanol (9) (CDCl 3). Results 107
Table 2.11 - NMR data of inuroyleanol (9) compared to the NMR data from the literature*. 9 Lit * 9 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.38 m 20, 2, 3, 10, 5, 9 1 36.4 t 36.5 β 3.30 m 3.26 dt (11 , 3.8) 3, 10 α 1.60 m 1.58 m 2 19.0 t 19.0 1.76 ddddd (13.8, β 1.74 m 13.8, 13.3, 3.5, 3.5) α 1.28 m 1.27 td (13.8 , 4.2) 2, 4, 18 3 41.1 t 41.2 β 1.51 m 1.49 dt (13 , 3.8) 2 4 33.4 s 33.4 4, 3, 18, 19, 10, 5 α 49.6 d 49.7 1.83 dd (10.7 , 6.5) 1.80 dd (10.7 , 6.2) 1, 20, 9, 6, 7 α 2.66 m 2.63 m 7, 8, 5, 10 6 35.8 t 35.9 β 2.63 m 2.63 m 7, 8, 5, 10 7 206.1 s 206.1 8 112.4 s 112.5 9 135.8 s 135.9 10 40.3 s 40.3 11 138.5 s 138.6 12 152.1 s 152.2 13 126.1 s 126.1 14 158.2 d 158.3 16, 17, 13, 12, 15 26.0 d 26.0 3.32 septet (7) 3.30 qq (7, 7) 14 16 20.2 q 20.3 1.41 d (7) 1.37 d (6.7) 13, 15 17 17 20.3 q 20.4 1.42 d (7) 1.39 d (6.7) 16, 13, 15 18 33.1 q 33.1 0.97 s 0.94 s 19, 4, 3, 5 19 21.5 q 21.6 0.98 s 0.96 s 18, 3, 4, 5 20 17.9 q 17.9 1.38 s 1.36 s 1, 5, 10, 9 CH O 3 62.0 q 3.81 s 3.78 s 12 at C 12 OH at 5.70 s 5.68 s 9, 11, 12 C11 OH at 13.40 s 13.36 s 8, 13, 14 C14 * (Fraga et al. 2005); **HMBC correlations are from proton (s) stated to the indicated carbon.
108 Results
2.3.2.10 Sugiol (10)
Figure 2.70 - Chemical structure of sugiol (10).
Isolation of compound 10 (11.9 mg) (Figure 2.70) is described in section 5.2.2. It was obtained as brown amorphous solid. The EI-MS spectrum (Figure 2.71) showed a molecular ion peak at m/z 300 [M] + and a fragmentation pattern similar to that of compound 8, but with
30 mass units less. Thus, the molecular formula C 20 H28 O2 was suggested. This is in agreement that both compounds differ in the presence of a methoxy group. Consequently, a signal for a methoxy group was missing in the NMR spectrum (see Figure 2.72 and Figure 2.73).
Moreover, the signal for C-11 was shifted upfield compared to compound 8 ( δC 144.0 to 1 109.9) and that one for C-9 downfield ( δC 145.4 to 156.4). The other signals in the H- and 13 C-NMR spectra showed similar chemical shifts as for compound 8. 1D and 2D NMR (see Figure 2.74, Figure 2.75, Figure 2.76 and Table 2.12) and MS analysis were in agreement with those reported for sugiol, a diterpene which has already been isolated from two Salvia species (Chang et al. 1990; Kolak et al. 2005). Hence, structure elucidation resulted in the identification of compound 10 as sugiol. Results 109
D:\icis\m fpbd58b 03/11/09 04:51:53 PM PL-8 EI-direkt s59 , Source:200',70eV,500uA ,m/z 41-600
m fpbd58b #1 RT: 2.17 AV: 1 NL: 1.02E7 T: + c EI Full ms [ 41.00-600.01] 285.2 10000000
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5000000 Intensity
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3500000 203.1 3000000 217.2
2500000 243.2
2000000 301.3 179.1 1500000 201.1 163.1 189.1 1000000 161.1 173.1 218.2 109.1145.1 159.1 244.2 500000 91.1 257.2 302.3 55.1 69.1 332.2 271.2 314.2 346.2 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 m /z
Figure 2.71 - EI-MS spectrum of sugiol (10).
110 Results
13 Figure 2.72 - C-NMR spectrum of sugiol (10) (100 MHz, CDCl 3).
Results 111
1 Figure 2.73 - H-NMR spectrum of sugiol (10) (400 MHz, CDCl 3).
112 Results ppm
Figure 2.74 - HMBC spectrum of sugiol (10) (CDCl 3).
Results 113 ppm 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 19 18 1.0 20 17 16 3 3 1.5 1 2 2 5 2.0 1 2.5 6 6 3.0 15 3.5 4.0 ppm 4.5 5.0 5.5 6.0 6.5 11 7.0 7.5 14 8.0 8 9 12 13 4 6 14 11 15 5 3 2 18 1/10 20 19 16/17
Figure 2.75 - HSQC spectrum of sugiol (10) (CDCl 3).
114 Results 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 0.8 18 1.0 19 16/17 1.2 20 3 1.4 3 1 1.6 2 2 1.8 5 2.0 ppm 2.2 1 2.4 2.6 6 6 2.8 3.0 15 3.2 1 2 15 5 6 1 6 3 2 16/17 20 3 18 19
Figure 2.76 - H;H-COSY spectrum of sugiol (10) (CDCl 3).
Results 115
Table 2.12- NMR data of sugiol (10) compared to the NMR data from the literature (*)(**). 10 Lit * 10 Lit ** 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC *** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.55 m 1.56 m 1 37.8 t 38.47 β 2.23 brd (12.8) 2.25 brdt (3, 13) α 1.68 m 2 18.8 t 19.39 1.79 ddddd (13.6, 13.6, β 13.6, 3.1, 3.1) α 1.30 m 18, 19, 4, 3 41.3 t 42.00 β 1.55 m 4 33.2 s 33.65 5 α 49.4 d 50.30 1.88 dd (13,5, 4.2) 1, 10, 20, 9 β 2.62 dd (13.5, 18) 2.52 dd (13.5, 17.5) 10, 5, 7 6 36.0 t 36.37 α 2.71 dd (18, 4.2) 2.64 dd (17.5, 3.5) 10, 5 7 7 198.6 s 197.50 8 124.6 s 124.34 9 156.4 s 158.61 10 37.8 s 38.47 11 109.9 d 110.19 6.73 s 6.70 s 10, 8, 13, 12 12 158.2 s 160.35 13 132.6 s 133.63 14 126.5 d 126.49 7.93 s 7.87 s 15, 7, 9, 12 15 26.7 d 27.24 3.17 septet (7) 3.20 sept (7) 16, 17, 12 16 22.3 q 22.54 1.26 d (7) 0.95 d (7) 13, 17, 15 17 22.4 q 22.86 1.27 d (7) 0.95 d (7) 13, 16, 15 18 32.5 q 32.61 0.94 s 1.22 s 19, 4, 3, 5 19 21.3 q 21.57 1.00 s 1.22 s 18, 4, 3, 5 20 23.2 q 23.41 1.23 s 1.22 s 9, 1, 10, 5
* (Chang et al. 1990); ** (Kolak et al. 2005); ***HMBC correlations are from proton (s) stated to the indicated carbon.
116 Results
2.3.2.11 Cryptojaponol (11)
O 16
12 15 17 HO 11 13
20
1 9 14 2 10 8
3 5 4 7 6 O
19 18
Figure 2.77 - Chemical structure of cryptojaponol (11).
Compound 11 (4 mg) (Figure 2.77) was isolated as described in section 5.2.2 and gave a brown amorphous solid. The EI-MS spectrum (Figure 2.78) showed the same fragmentation pattern as compound 8, but with different intensities. The molecular ion peak at m/z 330 [M] + is in agreement with the molecular formula C 21 H30 O3. 13 C-NMR data (Figure 2.79) only exhibited major differences in the chemical shifts for C- 9, C-12, C-13, and C-14 compared to compound 8, whereas the other shifts were similar. Therefore, it could be assumed that differences between both diterpenes may occur in the substitution at C-11 and C-12. The signal for the methoxy group at δH 3.83 exhibited a long- range correlation with C-12 ( δC 149.0) indicating its position at C-12 (see Figure 2.80 to Figure 2.83 and Table 2.13). This was in agreement with the upfield-shift compared to compound 8 which had a hydroxyl group at this position. The singlet for the hydroxy group at
δH 6.11 showed long-range correlations with C-9 ( δC 138.1), C-11 ( δC 146.5), and C-12 ( δC 149.0) confirming its location at C-11. Long-range correlations were observed between the singlet at δH 7.60 and the signals for C-7 ( δC 200.0), C-9 ( δC 138.1), C-12 ( δC 149.0), C-13 ( δC
139.0), and C-15 ( δC 26.6) which it is in agreement that C-14 is unsubstituted. Hence, according to the 1D and 2D 1H- and 13 C-NMR as well as MS data compound 11 was identified as cryptojaponol, an isomer of diterpene 8. Spectroscopic data from the literature were in line with this structure (Rodriguez 2003). Results 117
D:\icis\mfpbd87b 05/27/09 12:23:43 PM PE-13 EI-direkt s40, Source:200',70eV,500uA ,m/z 41-600 m fpbd87b #1 RT: 1.52 AV: 1 NL: 2.71E5 T: + c EI Full ms [ 41.00-600.01] 233.0 270000
260000
250000 245.0 240000
230000 330.1
220000
210000
200000
190000
180000 315.1 170000
160000
150000
140000
Intensity 130000
120000 219.0 110000 247.1
100000
90000
80000 331.1 70000 273.1 60000
50000 261.0 346.1 40000 193.0 30000 274.1 189.0 215.0 20000 173.0 148.9 283.0 69.0 83.0 127.9 347.1 10000 55.0 360.1 0 50 100 150 200 250 300 350 400 450 500 m /z
Figure 2.78 - EI-MS spectrum of cryptojaponol (11).
118 Results 10 20 2 20 19 16 17 15 30 4 18 6 1 10 40 3 5 50 12 60 at C at OCH3 3 70 CDCl 80 90 100 ppm 110 13 120 8 130 9 13 140 11 12 150 160 17 15 170 16 14 13 O 180 8 12 7 O 6 11 9 190 10 20 O 18 H 7 4 5 1 200 19 2 3
13 Figure 2.79 - C-NMR spectrum of cryptojaponol (11) (100 MHz, CDCl3). Results 119 0.5 1.0 1.5 18 19 1.0 2.0 1.2 2.5 16 17 3 6 2.5 2.6 1.3 6 2.7 1.4 20 3.0 3.15 15 3.24 3.5 1 1 1.5 3 12 3.33 1.6 at C at OCH3 2 4.0 1.7 ppm 2 1.8 4.5 5 1.9 5.0 5.5 6.0 11 C OH at 6.5 17 15 16 14 7.0 13 O 8 12 7 O 6 11 9 7.5 14 10 20 O 18 H 4 5 1 8.0 19 2 3
1 Figure 2.80 - H-NMR spectrum of cryptojaponol (11) (400 MHz, CDCl3).
120 Results ppm 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 0.5 18 1.0 19 16 17 3 20 1 3 1.5 2 2 5 2.0 2.5 6 6 3.0 15 1 3.5 12 C OCH3 at 4.0 ppm 4.5 5.0 5.5 11 6.0 OH at C 6.5 7.0 7.5 14 7 8 12 10 11 13 12 5 3 14 20 9 19 2 1 OCH3 at C 4 6 18 15 16/17
Figure 2.81 - HMBC spectrum of cryptojaponol (11) (CDCl 3). Results 121
ppm
Figure 2.82 - HSQC spectrum of cryptojaponol (11) (CDCl 3). 122 Results
Figure 2.83 - H;H-COSY spectrum of cryptojaponol (11) (CDCl 3). Results 123
Table 2.13 - NMR data of cryptojaponol (11) compared to the NMR data from the literature*. 11 Lit * 11 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] multiplicity J [Hz] α 1.47 m 1.39 ddd (13.6, 13.4, 3.5) - 1 35.6 t 36.19 β 3.26 m 3.23 ddd (13.6, 3.6, 1.2) - 1.57 ddddd (14, 3.8, 1.9, α 1.63 m - 3.5, 1.2) 2 18.9 t 18.95 1.75 dddt (14, 13.5, 3.3, β 1.77 m - 13.4, 3.6 ) α 1.27 m 1.26 ddd (13.7, 13.5, 3.8) 1, 4, 18 3 41.2 t 41.18 β 1.55 m 1.48 ddd (13.7, 3.3, 1.9) - 4 33.4 s 33.43 5 α 50.2 d 50.25 1.88 dd (13.4 , 4.1) 1.58 dd (12.4, 4.9) - α 2.68 dd (18 , 4.1) 2.64 dd (16.8) 5, 7 6 36.2 t 35.60 β 2.57 d d (18 , 13.6) 2.54 dd (16.8) 10, 7 7 200.0 s 199.22 8 128.8 s 128.79 9 138.1 s 138.06 10 40.1 s 40.16 11 146.5 s 146.46 12 149.0 s 149.07 13 139.0 s 139.04 14 117.3 d 117.25 7.60 s 7.60 s 15, 7, 9, 12, 13 15 26.6 d 26.61 3.21 septet (7) 3.18 sept (6.8) 14, 16, 17 16 23.5 q 23.54 1.25 d (7) 1.24 d (6.8) 15, 17, 13 17 23.4 q 23.46 1.25 d (7) 1.22 d (6.8) 15, 16, 13 18 33.1 q 33.11 0.96 s 0.92 s 19, 3, 4, 5 19 21.4 q 21.45 1.00 s 0.96 s 18, 3, 4, 5 20 18.0 q 17.95 1.40 s 1.38 s 10, 1, 5, 9 CH O 3 61.8 s 61.82 3.83 s 3.79 s 12 at C 12 OH at 6.11 s 6.15 s 9, 11, 12 C11
* (Rodriguez 2003); **HMBC correlations are from proton (s) stated to the indicated carbon.
124 Results
2.3.2.12 Orthosiphonol (12)
Figure 2.84 - Chemical structure of orthosiphonol (12).
Compound 12 (5.3 mg) (Figure 2.84) was isolated as described in section 5.2.2 and obtained as a light brown amorphous solid. The EI-MS spectrum (Figure 2.85) displayed a + molecular ion peak at m/z 346.1 [M] . The molecular formula C 21 H30 O4 was concluded from the ion at m/z 346. 21441 [M] + in the HREIMS. The 13 C-NMR (Figure 2.86) and 1H-NMR (Figure 2.87) spectra of 12 showed only minor differences in the chemical shifts compared to compound 9 suggesting that the chemical structures are very similar (see Table 2.14). Again, differences were only observed in the chemical shifts for C-9 ( δC 143.9), C-12 ( δC 155.6), C-
13 ( δC 119.8), and C-14 ( δC 161.8). One hydroxyl group was bound to C-12 and confirmed by long-range correlations between δH 6.40 and C-11 ( δC 137.2), C-12 ( δC 155.6), and C-13 ( δC
119.86). A long-range correlation was also observed between the three proton singlet at ( δH
3.75) and the signal for C-11 ( δC 137.2) and confirmed a methoxy group at C-11. Moreover, a singlet for a phenolic hydroxyl group appeared at δH 14.10. Long-range correlations between this signal and C-8, C-13, and C-14 indicated its position at C-14. The downfield shift of C- 14-OH can be explained by a hydrogen bond with the carbonyl at C-7 (see Figure 2.88, Figure 2.89 and Figure 2.90). Hence, compound 12 was identified as orthosiphonol, isomeric to compound 9. This diterpene has already been known from Orthosiphon wulfenioides (Xiang et al. 2002). Results 125
D:\icis\mfpbd86b 05/27/09 12:19:55 PM PE-12 EI-direkt s43, Source:200',70eV,500uA ,m/z 41-600 m fpbd86b #1 RT: 1.64 AV: 1 NL: 2.16E6 T: + c EI Full ms [ 41.00-600.01] 346.1
2100000
2000000
1900000
1800000
1700000
1600000
1500000
1400000 331.1
1300000
1200000
1100000 Intensity 1000000
900000
800000
700000
600000
500000 347.2 400000
300000 245.0 289.0 200000 219.0 249.0 271.0 313.1 217.0 100000 109.0 173.0 187.0 348.2 69.0 106.9 114.9 0 50 100 150 200 250 300 350 400 450 500 550 600 m /z
Figure 2.85 - EI-MS spectrum of orthosiphonol (12).
126 Results 10 20 30 40 5 50 11 60 at C at OCH3 70 80 2 16 20 17 90 19 20 15 100 25 8 110 ppm 30 13 120 18 130 35 6 11 1 140 9 4 40 10 3 150 12 160 14 17 170 15 16 OH 14 180 13 O 8 12 OH 7 190 6 11 9 10 5 20 O 200 18 7 1 4 19 210 2 3
13 Figure 2.86 - C-NMR spectrum of orthosiphonol (12) (100 MHz, CDCl3). Results 127
1 Figure 2.87 - H-NMR spectrum of orthosiphonol (12) (400 MHz, CDCl3).
128 Results ppm
Figure 2.88 - HMBC spectrum of orthosiphonol (12) (CDCl 3). Results 129 10 15 20 25 30 35 40 45 50 55 60 65 0.4 0.6 0.8 1.0 19 18 1.2 3 1.4 20 17 3 16 1.6 1 2 2 5 1.8 2.0 ppm 2.2 2.4 2.6 6 6 2.8 1 3.0 3.2 3.4 15 11 3.6 C OCH3 at 3.8 4.0 4 10 8 18 15 3 11 2 1 19 6 20 16/17 OCH3 at C
Figure 2.89 - HSQC spectrum of orthosiphonol (12) (CDCl 3).
130 Results ppm
Figure 2.90 - H;H-COSY spectrum of orthosiphonol (12) (CDCl 3). Results 131
Table 2.14 - NMR data of orthosiphonol (12) compared to the NMR data from the literature*. 12 Lit * 12 Lit * 13 C δ 13 C δ 1H δ/ppm, 1H δ/ppm, Position HMBC ** /ppm /ppm multiplicity J [Hz] Multiplicity J [Hz] α 1.65 m 1.65 m 10, 3 1 37.4 t 37.45 2.93 ddd (12.8, 3.2, β 2.94 m 1.7) α 1.65 m 1.65 m 2 19.1 t 19.14 β 1.75 m 1.66 m 3, 4, 10 α 1.30 m 1.34 m 3 40.9 t 40.95 β 1.50 m 1.50 m 2 4 39.7 s 33.53 3, 4, 6, 7, 9, 10, 5 α 49.1 d 49.18 1.81 dd (8.7, 8.7) 1.82 dd (3.7, 11.5) 18, 19, 20 α 2.63 m 2.65 m 4, 5, 7, 10 6 35.6 t 35.60 β 2.66 m 2.65 m 4, 5, 7, 10 7 204.4 s 204.44 8 108.8 s 108.85 9 143.9 s 144.00 10 40.3 s 40.38 11 137.2 s 137.25 12 155.6 s 155.70 13 119.8 s 119.86 14 161.8 d 161.87 15 24.2 d 24.21 3.50 septet (7) 3.16 h (7.1) 12, 13, 14, 16, 17 16 20.0 q 20.07 1.33 d (7) 1.34 d (7.1) 13, 15, 17 17 20.1 q 20.16 1.35 d (7) 1.35 d (7.1) 13, 15, 16 18 33.5 q 33.00 0.95 s 0.96 s 3, 4, 5, 19 19 21.6 q 21.69 0.99 s 1.00 s 3, 4, 5, 18 20 21.2 q 21.23 1.38 s 1.36 s 1, 5, 9, 10 CH O 3 61.9 s 61.80 3.75 s 3.75 s 11 at C 11 OH at 6.40 s 6.46 brs 11, 12, 13 C12 OH at 14.10 s 13.30 brs 8, 13, 14 C14
* (Xiang et al. 2002); **HMBC correlations are from proton (s) stated to the indicated carbon.
132 Results
2.3.3. HPLC analysis
HPLC analysis of the n-hexane extract of P. longipes was performed, and all isolated diterpenes were assigned to their respective peak in the chromatogram (Figure 2.91). Quantification using a calibration curve with the respective isolated compound revealed that 7α-acetoxyroyleanone ( 1) was the main diterpene in the n-hexane extract with a concentration of 23.4%.
A mAU 1 1600
1400
1200
1000
800
600
400
2 12 200 5 3 6 8 11 9 4 10 7 0
5 10 15 20 min 25
Figure 2.91 - HPLC chromatogram of the n-hexane extract from Peltodon longipes prepared from the roots (injection: 10 µµµl = 5 µµµg extract), concentrations of the main compound: 1: 24.3%. Further HPLC conditions are given in the Experimental Section 5.2.1.
Results 133
2.3.4. Biological studies
2.3.4.1 Cytotoxic studies of diterpenes from P. longipes and from Salvia species using the MTT assay
The cytotoxic effects of the n-hexane extract of Peltodon longipes as well as the isolated diterpenes 1 - 12 were studied in two selected cancer cell lines, MIA PaCa-2 and MV-3 as described in section 5.2.3.4. At first, MIA PaCa-2 cells were treated with different concentrations of diterpene 1 for 1h, 2h, 16h, 24h and 48h and cytotoxic effects were evaluated using the MTT assay. As shown in Figure 2.92, the cytotoxic activity was concentration and time dependent. For determination of the IC 50 values of the other diterpenes in MIA PaCa-2 cells an incubation time of 24h was chosen. The same conditions were used in MV-3 cells (Table 2.15). Diterpenes 1, 2, 4, 6, 8 and 10 exhibited cytotoxic activity in both cell lines. Compound 1 was the most active with an IC 50 value of 4.7 µM in MIA PaCa-2 cells and of 7.4 µM in MV-3 cells. Compound 333 only showed a moderate activity in the pancreatic cell line, whereas 5, 7, 9, 11 and 12 gave IC 50 values > 100 µM in both cell lines. MIA PaCa-2 cells were mostly more sensitive to the compounds. Camptothecin (CPT) was used as a positive control.
7α-acetoxyroyleanone
120
100
1h 80 2h 16h 60
viability [%] 24h 48h 40
20
0 0.5 1 2 4 8 16 concentration [µM]
Figure 2.92 - Cell death (%) after different exposition time in MIA PaCa-2 cells with 0.5 to 16 µM of 7 α-acetoxyroyleanone (1) evaluated by MTT assay.
134 Results
Table 2.15 - Cytotoxic activity of the diterpenes 1 - 12 isolated from Peltodon longipes and 13 - 21 from Salvia species in MIA PaCa-2, a human pancreatic carcinoma cell line and MV-3, a human melanoma cancer cell line using the MTT assay. Data are presented as IC 50 values and 95% confidence interval from three independent experiments. Camptothecin was used as positive control.
Cell line IC 50 (µM) Compound/extract MIA PaCa-2 MV-3 Peltodon longipes - extract* 1.3* (1.2 – 1.4) 2.9 (2.8 – 3.1)
7α-acetoxyroyleanone ( 1) 4.7 (4.4 – 5.1) 7.4 (4.9 – 11.1)
Horminone ( 2) 27.5 (25.5 – 29.7) 16.7 (14.7 – 18.9)
Royleanone ( 3) 32.5 (29.2 – 36.0) > 80
7-ketoroyleanone ( 4) 30.1 (28.2 – 31.2) 65.8 (61.0 – 70.8)
7α-ethoxyroyleanone ( 5) > 100 > 80
Iguestol ( 6) 41.3 (34.5 – 46.8) 65.9 (60.4 – 71.9)
Deoxyneocryptotanshinone ( 7) > 100 > 150 12-hydroxy-11-metoxyabieta-8,11,13- 34.9 (29.7 – 41.0) 32.3 (30.4 – 34.3) trien-7-one ( 8) Inuroyleanol ( 9) > 80 > 120
Sugiol ( 10 ) 17.9 (15.6 – 20.6) 34.1 (30.0 – 39.1)
Cryptojapanol ( 11 ) > 100 > 80
Orthosiphonol ( 12 ) > 100 > 80
Salvia miltiorrhiza - extract* 1.8* (1.5 – 2.3) nd
Tanshinone IIa ( 13 ) 1.9 (1.6 – 2.3) nd
Cryptotanshinone ( 14 ) 5.8 (4.9 – 6.9) nd
Tanshinone I ( 15 ) 10.5 (9.1 – 12.1) nd
1,2-Dihydrotanshinone I (16 ) 5.6 (4.7 – 6.7) nd
Miltirone ( 17 ) 22.5 (21.7 – 23.5) nd
1-oxomiltirone ( 18 ) 29.9 (26.4 – 33.8) nd
Miltiodiol ( 19 ) 66.3 (54.9 – 80.1) nd
Ferruginol ( 20 ) 25.9 (23.5 – 28.7) nd
Sahandinone ( 21 ) 10.2 (9.1 – 11.4) nd
Camptothecin 0.4 (0.3 – 0.5) nd
* IC 50 expressed in µg/ml; nd not determined. Results 135
Several diterpenes from P. longipes possess a para -naphthoquinone skeleton. To compare the effect of ortho and para -quinone moieties on cytotoxicity six 20-nor-abietanes ( 13 - 18 ), one secoabietane diterpene ( 21 ) with an ortho -naphthoquinone structure, and two diterpenes lacking a quinone structure ( 19 - 20 ) were included in the cytotoxicity study using MIA PaCa- 2 cells. These compounds had been previously isolated from roots of Salvia miltiorrhiza (Slusarczyk et al. 2011) and S. sahendica (compounds 20 and 21 ) (Jassbi et al. 2006b) (for structures see Figure 2.93). Tanshinone IIa ( 13 ), an abietane with a furan ring, exhibited the highest cytotoxicity in MIA PaCa-2 cells (IC 50 1.9 µM). Compounds bearing a dihydrofuran ring ( 14 ) or additional double bonds in ring A ( 15 and 16 ) were less cytotoxic. An isopropyl moiety instead of the heterocyclic D-ring further lowered activity (IC 50 of 22.5 and 29.9 µM for 17 and 18 , respectively). The monohydroxy derivative 20 gave a similar IC 50 value of 25.9 µM. Cytotoxic activity dramatically decreased with the dihydroxy derivative 19 which additionally has a carbonyl group (IC 50 66.3 µM). Interestingly, the secoabietane 21 had an
IC 50 of 10.2 µM, although missing an intact ring A.
OH OH OH OH O OH
O O O O HO O
O O O O O
O OH O O O OH 1 2 3 4 5 6
OH O OH OH OH O
O HO O O HO
OH OH O
O O O O O
7 8 9 10 11 12
O O O O O
O O O O O
O O O O
13 14 15 16 17
O OH OH
O HO
O O O
O 21 18 19 20
Figure 2.93 - Chemical structures of the diterpenes isolated from Peltodon longipes (1 - 12), Salvia miltiorrhiza (13 - 19) and Salvia sahendica (20 and 21). 136 Results
Cytotoxic studies of diterpene 1 using FACS analysis and PI staining The MTT test is widely used to measure cell cytotoxicity as well as proliferation or viability. However, little attention has been given to the ability of extracellular factors that can reduce MTT and lead to false negative cytotoxic results. Thus, reduction of MTT by herbal extracts or natural compounds in the absence of cells have been reported (Shoemaker et al. 2004; Bruggisser et al. 2002). To confirm the cytotoxicity observed in the MTT assay, 7α- acetoxyroyleanone ( 1) was exemplarily studied by FACS analysis after DNA staining with propidium iodide (PI), which can not enter in living cells and detects dead cells. The IC 50 value obtained for compound 1 of 4.3 µM (3.7 - 5.0) using MIA PaCa-2 cells was in line with that one from the MTT assay (IC 50 of 4.7 µM) (see Figure 2.94).
Control ( 1) 2 µM ( 1) 4 µM ( 1) 8 µM
Gate M1 (cell death) 15.84% 40.06% 81.20%
Figure 2.94 - Cytotoxic activity of 7 α-acetoxyroyleanone (1) evaluated by FACS analysis after DNA staining with PI. MIA PaCa-2 cells were treated with the same conditions as in the MTT assay.
Cytotoxic studies of diterpenes 1 and 3 in the NCI cell panel 7α-acetoxyroyleanone ( 1) and royleanone ( 3) were also screened against the NCI-60 cell line panel. The compounds were initially tested at a single dose of 10 µM (see Table 2.16 and Table 2.17). Compound 1 was further evaluated against the same NCI-60 panel at five concentrations (0.01 - 100 µM). The results were reported as a mean graph of the GI 50 (which measures the growth inhibitory power of the tested agent), TGI 50 (represents the cytostatic effects) and the LC 50 (represents the cytotoxic effects). 7α-acetoxyroyleanone ( 1) exhibited cytotoxic effects with remarkable differences in specificity depending on the cell line studied.
As shown in Table 2.18, an LC 50 of about 7.4 µM was observed in nine melanoma cell lines, whereas in six different leukemia cells the LC 50 could not be determined (>100 µM). The NCI in vitro anticancer screening program generates prodigious quantities of information-laden biological test results, which can be captured in an easily accessible Results 137 computerized database. This database can be used by the program COMPARE in order to find similar compounds to the respective tested compound and to predict the biochemical mechanism of action. Similarity is quantitatively expressed as a Pearson Correlation Coefficient (PCC) (Shoemaker 2006). The results obtained with the COMPARE algorithm program for compound 1 did not show any match with a high correlation. The maximum PCC was 0.645 when using 30 common cell lines as a prerequisite for the analysis. 138 Results
Table 2.16 - Effect of 7 α-acetoxyroyleanone (1) at a 10 µM concentration on NCI 60 human solid tumor cells represented as growth percentage.
Results 139
Table 2.17 - Effect of royleanone (3) at a 10 µM concentration on NCI 60 human solid tumor cells represented as growth percentage.
140 Results
Table 2.18 - Effect of 7 α-acetoxyroyleanone (1) against the 60 cell line panel at five concentrations. Results are shown as GI 50 (growth inhibitory), TGI 50 (cytostatic effect) and LC 50 (cytotoxic effect).
Results 141
2.3.4.2 Studies on the alkylating properties of compounds 1 - 4 and 10
Quinones cytotoxicity is often explained by their ability to covalently bind to proteins, DNA, and RNA (Begleiter 1985; Bolton et al. 2000; Hasinoff et al. 2005). Therefore, diterpenes 1 - 4 differing in their substitution at C-7, and 10 , in which the para -quinone system was replaced by an aromatic ring, were studied for their alkylating properties in an extracellular system using 4-(4-nitrobenzyl)pyridine (NBP) (Bardos et al. 1965; Thomas et al. 1992). This nucleophile is a trap for alkylating agents with nucleophilic characteristics similar to DNA bases. Diterpenes 1 - 3 exhibited a similar reactivity towards NBP, whereas 4 and 10 revealed no alkylating properties under these conditions (see Figure 2.95). The different behavior of compounds 1, 4 and 10 can be explained in such a way, that only diterpenes 1, 2 and 3 can react with NBP (see scheme Figure 2.96), but not diterpenes 4 and 10 . p- Benzoquinone was used as positive control and showed the highest reactivity. These results indicate that cytotoxicity of 1 - 3 does not correlate with their alkylating properties. Otherwise compound 1 should show the highest reactivity. Moreover, cell killing of 4 and 10 must be explained by another mechanism.
7 α-acetoxyroyleanone (1) 1.2 horminone (2)
royleanone (3) 0.9 sugiol (10 )
7-ketoryleanone (4) 0.6 p-benzoquinone
absorbance at 540nm 0.3
0.0 0.0 25.0 50.0 75.0
concentration [µM]
Figure 2.95 - Comparative reaction rates of diterpenes 1 - 4 and 10 at various concentrations with NBP at 37°C (for detailed information see section 5.2.3.8). p-Benzoquinone was used as positive control. Data are mean (± SD) of three independent experiments .
142 Results
OH O
O N +O OH N - O OH OH - O O
O O + + N H N
- HO
+ N - + O N - O O O
OH OH HO OH HO HO O O + - O N HO + N H N H
+ N + + N - O N O - - - O O O O
coloured adduct
Figure 2.96 - Proposed chemical reaction of the diterpene 2 with NBP.
In addition, the alkylating properties of diterpene 1 were also determined in the presence of the reducing agent DTT (Begleiter et al. 1991). The results presented in Figure 2.97 revealed no differences in the alkylating properties of the reduced form of compound 1. Results 143
0.4 w ith DTT
w ithout DTT
0.3
0.2 absorbance540nm at 0.1
0.0 0.0 25.0 50.0 concentration [µM]
Figure 2.97 - Reaction rates of diterpene 1 at various concentrations with NBP at 37°C (for detailed information see section 5.2.3.8) in the presence ( •••) or absence ( x) of 10 mM concentration of DTT. Data are mean (± SD) of two independent experiments .
2.3.4.3 Studies on human DNA topoisomerase I and II inhibitory activity
p-Quinone derivatives including 7-ketoroyleanone (diterpene 4) and quinolone derivatives are reported to intercalate with the DNA by targeting topoisomerases I or II (Han et al. 2008; Lindsey et al. 2004; Hawtin et al. 2010). These results raise the question of whether inhibition of topoisomerases may also be involved in the described cytotoxicity of diterpenes 1 - 4 and 10 , and whether there are preferences towards topoisomerase I or II. At first, the ability of the five diterpenes to inhibit topoisomerase I relaxation activity was studied at different concentrations from 1 nM to 1 mM. Camptothecin, the classical topo I inhibitor, was used as positive control (see Figure 2.98). As shown in Figure 2.98 and Table 2.19, all diterpenes significantly inhibited human topoisomerase I relaxation activity. Compounds 4 and 10 were the most active with average IC 50 values of 2.8 and 4.7 M, respectively, and even more active as the control camptothecin, which had an average IC 50 value of 28.0 M. Compound 1 was slightly less active with an IC 50 of 14.2 M similar to that of compound 3 with an average
IC 50 of 16.2 M. The less active compound was 2, although this still exhibited significant activity against topoisomerase I with an average IC50 of 32.8 M, which was comparable to camptothecin. 144 Results
camptothecin - 0 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14
CPT 7ααα-acetoxyroyleanone (1)
- 0 20 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CPT horminone (2)
- 0 20 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CPT royleanone (3)
- 0 20 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CPT 7- ketoroyleanone (4)
- 0 20 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CPT sugiol (10)
- 0 20 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 2.98 - Concentration dependent effects of diterpenes 1 - 4 and 10 on topoisomerase I mediated relaxation of pBR322 DNA. Agarose gels stained with ethidium bromide are shown. The conversion of relaxed plasmid DNA (R) to supercoiled molecules (S) was evaluated. Control reactions were carried out in the absence of diterpene and enzyme (labelled as minus -) or in the absence of diterpene but containing enzyme (labelled 0). Topoisomerase I concentration was constant. Camptothecin (CPT) was used as positive control. Data were consistent of two independent experiments. Results 145
Table 2.19 - Inhibitory effects of diterpenes 1 - 4 and 10 on human DNA topoisomerase I and II (IC 50 inhibition rate of relaxation). IC 50 values (µM) and the standard error of the mean ± S.E.M. for n = 2 are given.
IC 50 (µM) Compound Topo I Topo II 7α-acetoxyroyleanone ( 1) 14.2 ± 2.3 nd horminone ( 2) 32.8 ± 3.4 nd royleanone ( 3) 16.2 ± 2.7 nd 7-ketoroyleanone ( 4) 2.8 ± 0.5 26.7 ± 2.2 sugiol ( 10 ) 4.7 ± 0.2 26.0 ± 3.1 camptothecin (CPT) 28.0 ± 0.8 nd etoposide (ETO) nd 58.2 ± 0.2 nd = not determined
To find out if these diterpenes selectively inhibit topoisomerase I relaxation activity the two most active diterpenes 4 and 10 were also tested towards topoisomerase II in the same experimental setting (see Figure 2.99). Both compounds exhibited similar inhibitory activity against topoisomerase II with average IC 50 values of 26.7 µM and 26.0 µM, respectively.
When compared to etoposide, which had an average IC50 of 58.2 µM, both diterpenes were found to have an approximately 2-fold increase in inhibitory activity. Comparing the IC 50 values with those obtained from inhibition of topoisomerase I, it can be concluded that these diterpenes preferentially inhibit topoisomerase I under these conditions. 146 Results
etoposide
- 0 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14
ETO 7- ketoroyleanone (4)
- 0 50 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
ETO sugiol (10)
- 0 50 100 0.01 0.05 0.1 0.5 1 5 10 20 50 100 500 1000 M
R.-
S.- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 2.99 - Concentration dependent effect of diterpenes 4 and 10 on topoisomerase II mediated relaxation of pBR322 DNA.
Agarose gels stained with ethidium bromide are shown. The conversion of relaxed plasmid DNA (R) to supercoiled molecules (S) was evaluated. Control reactions were carried out in the absence of diterpene and enzyme (labeled as minus -) or in the absence of diterpene but containing enzyme (labelled 0). Topoisomerase II concentration was constant. Etoposide (ETO) was used as positive control. Data were consistent in two independent experiments
2.3.4.4 Molecular modeling studies
Inhibitors can differently target topoisomerase I and II leading to DNA damage and finally to cell death (Salerno et al. 2010). Diterpenes 1 - 4 and 10 were shown to inhibit topoisomerase I even at lower concentrations than the well-known camptothecin. This alkaloid is known to intercalate in the DNA because of its flat structure, mimic a nucleotide and prevent relegation of the single strand break induced by topoisomerase I (Lauria et al. 2007; Staker et al. 2005). To elucidate whether diterpenes 1 - 4 and 10 acts in a similar way as camptothecin they were docked into the binding pocket characteristic for camptothecin using the Induced Fit protocol of Glide. The resulting docking score of CPT was conspicuously Results 147 lower (-10.69) compared to the scores of the diterpenes (-7.05 to -5.45) indicating a higher binding affinity of camptothecin at the topoisomerase I DNA cleavage site. Accordingly, docking scores did also not correlate with the experimentally determined IC 50 values for inhibition of topoisomerase I. In contrast to CPT, the non-planar conformation of the diterpenes makes an intercalation difficult within the cleaved site of the DNA as observed for camptothecin (see Figure 2.100). Subsequently, alternative docking modes were evaluated using the DNA-unbound form of topoisomerase I. Potential binding pockets were determined by FTMAP algorithm (Brenke et al. 2009). Based on Fourier domain correlation techniques the algorithm identified five different pockets located at the surface of the DNA-interaction site that come into question for the binding of potential DNA competitive inhibitors. The diterpenes were docked into the proposed pockets using the Glide QM-Polarized Ligand Docking protocol (Friesner et al. 2004; Halgren et al. 2004). One of these binding pockets had significantly lower docking-scores compared to the other four pockets and exhibited a correlation with the experimentally determined IC 50 values (Table 2.20). These results proposed a direct interaction of the diterpenes with topoisomerase I in a new described binding mode (binding pocket: Arg-488, Gly-490, Asn-491, Lys-493, Thr-501). However, diterpenes 1 - 3 may also interact with the DNA, thus preventing the enzyme-DNA binding because of their proven alkylating properties.
Table 2.20 - Virtual binding affinity to topoisomerase I determined by FTMAP algorithm for diterpenes 1 - 4 and 10 with the respective inhibitory effects on DNA topoisomerase I (IC 50 inhibition rate of relaxation). Topo I G score Compound Relaxation assay (Kcal/mol) IC 50 (µM) 7α-acetoxyroyleanone ( 1) 14.2 ± 2.3 -6.92 horminone ( 2) 32.8 ± 3.4 -6.55 royleanone ( 3) 16.2 ± 2.7 -5.85 7-ketoroyleanone ( 4) 2.8 ± 0.5 -8.3 sugiol ( 10 ) 4.7 ± 0.2 -8.25 camptothecin (CPT) 28.0 ± 0.8 -
148 Results
A B
(A)
C D
Figure 2.100 - Docking approach of camptothecin and diterpenes to topoisomerase I. (A) shows the predicted binding mode of camptothecin, (B) diterpene 1 in the topoisomerase I-DNA covalent complex. In (C) the diterpenes 1 - 4 and 10 are docked in the binding pocket. (D) shows that binding of diterpene 1 in this pocket prevents the building of the topoisomerase I-DNA complex.
2.3.4.5 Cell cycle analysis
Inhibition of topoisomerases can lead to DNA damage which the cell tries to repair. Efficient DNA repair requires cell cycle arrest. As diterpenes 1 - 4 and 10 were shown to inhibit topoisomerases they were also studied on their influence on cell cycle progression in MIA PaCa-2. The percentage of cells in each phase of the cell cycle (G0/G1, S and G2/M) was determined after 24h treatment. As shown in Figure 2.101, the majority of the control cells exposed to DMSO were in the G0/G1 phase of the cell cycle and only a small percentage Results 149 was detected in either the S or G2/M phase. Diterpene 1 significantly increased the proportion of cells in the S phase at a 1 µM concentration ( p<0.01), and decreased the percentage of cells in the G0/G1 phase. This effect was even more significant ( p<0.001) when the cells were treated with a concentration of 2 µM, where 37.3% cells were found in the S phase, compared to 5.2% for untreated cells. In contrast, diterpenes 4 and 10 increased the proportion of cells in the G0/G1 phase, while the percentage of cells was mainly decreased in the S phase with a slight decrease in the G2/M phase. After 24h treatment with 30 µM 4 exhibited a significant (p<0.05) increase in the G0/G1 phase (70.3% vs 53.5% control). Compound 10 showed similar results with a significant ( p<0.01) increase in the G0/G1 phase (80.3% vs 53.5% control) and significant ( p<0.05) decrease in the S phase (10.2% vs 30.9% control) at a 20 µM concentration. Compounds 2 and 3 did not significantly influence the cell cycle distribution up to a tested concentration of 40 µM.
150 Results
7α-acetoxyroyleanone G0/G1
90 S 80 G2/M 70 60 b b 50 c c A 40 b 30 20 cellcycle distribution [%] 10 0 control 0.5 1 2 4 Concentration [µM]
90 7 Ketoroyleanone 80 a
70 60
50
B 40 30 20 a cellcycle distribution [%] 10 0 control 5 10 20 30 Concentration [µM] 90 b Sugiol 80
70
60
50
C 40
30
cell cycle distribution [%] cycle cell distribution 20 a
10
0 control 5 10 20 Concentration [µM]
Figure 2.101 - Effect of diterpene 1 (7 ααα-acetoxyroyleanone) (A), 4 (7 ketoroyleanone) (B) and 10 (sugiol) (C) on cell cycle progression. MIA PaCa-2 cells were treated with increasing concentrations of each compound for 24h and then the cell cycle distribution in each phase was analysed by flow cytometry. Data are mean (± S.E.M.) of three independent experiments. Statistic analysis was calculated using the Student’s t-test at a significant level of a p < 0.05, b p < 0.01, c p < 0.001, respectively compared to the control.
Results 151
2.3.4.6 Apoptotic activity using caspase-3 like assay
The ability of diterpenes to inhibit human DNA topoisomerase and disturb the cell cycle progression prompted us to investigate whether the diterpenes 1 - 4 and 10 influence activation of effector caspase-3/7 in MIA PaCa-2 cells using the fluorogenic DEVDase assay (for details see 5.2.3.5). The results are expressed as relative fluorescence units (RFU) referred to untreated control. The extract from Peltodon longipes was also included in the study. At first, MIA PaCa-2 cells were treated at different time points and with different concentrations of the n-hexane extract of P. longipes and 7 α-acetoxyroyleanone ( 1) (see Figure 2.102 and Figure 2.103). The crude extract and the compound 1 induce apoptotic cell death in a dose and time dependent manner.
10h 16h 16 24h
13.9
14 13.2
12
9.7 9.6 10 9.1
RFU 8.4 8 6.6 6.1 6 5.8 5.7 5.2
4.4 4.0 3.8 3.7 3.7 3.8 4 3.4
2
0 1 µg/ml 2 µg/ml 5 µg/ml 10 µg/ml ActD control 5 µg/ml Peltodon longipes
Figure 2.102 - Study of the effect on caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 10, 16 and 24h with the indicated concentrations of the n- hexane extract of P. longipes. Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments.
152 Results
10h 16h
16 24h 13.9
14 12.8
12 11.5
10 9.1 8.6 8.9 8.5 RFU 8 7.5 7.6
6.0 5.7 6 5.4 5.2 4.7 4.9 3.9 3.8 4 3.4
2
0 1 µM 2 µM 4 µM 8 µM ActD Control 5 µg/ml 7 α-acetoxyroyleanone ( 1)
Figure 2.103 - Effect of caspase-3/7 using the fluorogenic caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 10, 16 and 24h with the indicated concentrations of 7 α-acetoxyroyleanone (1). Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments.
Additionally, horminone ( 2), royleanone ( 3), 7-ketoroyleanone ( 4) and sugiol ( 10 ) were tested for their ability to activate procaspase-3/7. MIA PaCa-2 cells were exposed for 24h with the diterpenes at three different concentrations as observed in Figure 2.104. None of the compounds induced a significant level of apoptotic cell death.
Results 153
20.0
15.0 13.9
9.8 10.0 8.8 RFU RFU 7.1 6.3 6.1 5.7 6.2 5.7 5.3 5.7 5.4 5.1 5.0 4.1
0.0 CTRL ActD (2) (2) (2) (3) (3) (3) (4) (4) (4) (10) (10) (10) 5µg/ml 10µM 20µM 40µM 10µM 20µM 40µM 20µM 40µM 80µM 20µM 40µM 80µM
Figure 2.104 - Effect on caspase-3/7 using the fluorogenic caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 24h with the indicated concentrations of horminone (2), royleanone (3), 7-ketoroyleanone (4) and sugiol (10). Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments.
2.3.4.7 Cell death detection ELISA
7α-Acetoxyroyleanone ( 1), horminone ( 2) and royleanone ( 3) as well as the n-hexane extract of P. longipes were also studied for their apoptotic effects in the cell death ELISA plus Kit, which quantifies apoptosis-associated DNA fragmentation. MIA PaCa-2 cells were exposed for 16h with different concentrations of the compounds or the extract. The effects expressed as enrichment factor were only moderate and mostly observed at high concentrations (see Figure 2.105). Therefore, only one experiment was performed. 154 Results
2.5
2.1 2 1.8
1.6 1.5 1.5 1.3
1.1 1.1 1.0 1
enrichment factor enrichment 0.8 0.7
0.5
0 control ActD 5 µg/ml 10µg/ml 4 µM 8 µM 40 µM 80 µM 40 µM 80 µM 5µg/ml Extract P. longipes 7α−acetoxyroyleanone horminone royleanone
Figure 2.105 - Effects on apoptosis-associated DNA fragmentation using the cell death detection ELISA plus Kit (depicted as enrichment factor) after 16h exposure in MIA PaCa-2 cells with the indicated concentrations of the n-hexane extract of Peltodon longipes , 7 α-acetoxyroyleanone (1), horminone (2) and royleanone (3). Actinomycin D (ActD) was used as positive control.
2.3.4.8 Apoptotic cell death measured by flow cytometry
Furthermore, internucleosomal DNA fragmentation as a characteristic feature for apoptosis was measured on the single cell level by flow cytometry and the percentage of cells with a sub-G1 DNA content was determined. Again, apoptotic effects were most pronounced with diterpene 1 and comparable with that of camptothecin. Diterpene 2 showed a similar effect only at higher concentrations, whereas the effect of diterpenes 3, 4 and 10 was only very slight and in agreement with the low effect on caspase 3/7. Results 155
30.0
25.0 23.1 23.4 21.4 20.1 20.0 18.6
15.0 13.9 11.9 10.8
10.0 8.7 % of apoptotic cell death cell %apoptotic of 5.5 5.0 5.0 5.1 4.1 5.0 3.2 2.9 3.2 2.0
0.0 CTRL CPT CPT (1) (1) (1) (2) (2) (2) (3) (3) (3) (4) (4) (4) (10) (10) (10) 1µM 10µM 2µM 4µM 8µM 10µM 20µM 40µM 10µM 20µM 40µM 10µM 20µM 30µM 5µM 10µM 20µM
Figure 2.106 - Apoptotic cells measured as alteration in the “sub-G1” DNA content after 24h treatment with different concentrations of diterpenes 1 - 4 and 10. Camptothecin (CPT) was used as positive control.
2.3.4.9 Protein kinase inhibitory activity
Since the diterpenes have been shown to influence the cell cycle progression, the effects of 7α-acetoxyroyleanone and sugiol were investigated on their inhibitory effects towards nine key regulators of cell cycle protein kinases. The IC 50 values for the tested compounds in the protein kinase assay are presented in Table 2.21. The IC 50 calculated for 7 α- acetoxyroyleanone ( 1) showed inhibition values ranging from 3.0 x 10 -5 M to >1 x 10 -4 M.
The strongest inhibition was found for CDK7CycH/MAT1 and CDK8/CycC with an IC 50 value of 3.0 x 10 -5 M (30 µM). Sugiol ( 10 ) exhibited a slight inhibitory activity under the -5 -4 tested conditions with IC 50 values ranging from of 9.6 x 10 M to >1 x 10 M.
156 Results
Table 2.21 - Effect of 7 ααα-acetoxyroyleanone and sugiol on nine protein kinase of the cell cycle 33 ® evaluated in the radiometric protein kinase assay ( PanQinase Activity Assay) IC 50 values are given in Molar (M).
Compound Kinase IC 50 (M) CDK1/CycB1 3.9 x 10 -5 CDK2/CycE 6.0 x 10 -5 CDK3/CycE 5.4 x 10 -5 CDK4/CycD3 4.6 x 10 -5 1 (7α-acetoxyroyleanone) CDK6/CycD1 3.2 x 10 -5 CDK7/CycH/MAT1 3.0 x 10 -5 CDK8/CycC 3.0 x 10 -5 CDK9/CycT 4.7 x 10 -5 PCTAIRE1 4.0 x 10 -5 CDK1/CycB1 > 1 x 10 -4 CDK2/CycE > 1 x 10 -4 CDK3/CycE 9.8 x 10 -5 CDK4/CycD3 9.6 x 10 -5 10 (sugiol) CDK6/CycD1 9.9 x 10 -5 CDK7/CycH/MAT1 > 1 x 10 -4 CDK8/CycC > 1 x 10 -4 CDK9/CycT > 1 x 10 -4 PCTAIRE1 > 1 x 10 -4
2.3.4.10 Determination of DNA damage and repair using the alkaline comet assay
As described above, para -quinone abietane diterpenes ( 1 - 4 and 10 ) are inhibitors of topoisomerases I and II for which DNA damage is often reported (Lauria et al. 2007). Interestingly, previous results also demonstrated that the para -quinone abietane diterpenes 1 - 3 may target directly the DNA, leading to DNA damage in colonic and hepatic human cells (Slamenova et al. 2004). It was discussed that the observed DNA strand breaks originate from Results 157 alkali-labile sites, as they only occurred under alkaline conditions. Considering these contrasting reports 7 α-acetoxyroyleanone ( 1), which possesses alkylating properties, and sugiol ( 10 ), without alkylating properties, were tested for their ability to induce DNA single- strand and double-strand breaks in MIA PaCa-2 cells. As a suitable tool the comet assay was used, which is based on the ability tomeasure DNA strand breaks that caused relaxation of DNA supercoilds. A weak electric field stretches out the relaxed DNA in the direction of the anode which appears as a tail of a comet in the fluorescence microscope. As observed in Figure 2.107 after 1h exposure 7 α-acetoxyroyleanone ( 1) and sugiol ( 10 ) induced a dose- dependent DNA damage indicated by the olive tail moment (OTM) which corresponds to the smallest detectable size of migrating DNA (comet tail length) and the number of relaxed/broken pieces (represented by the intensity of DNA in the tail). 7 α-acetoxyroyleanone (1) (Figure 2.107 A) induced a significant damage at 2 µM concentration, and sugiol ( 10 ) (Figure 2.107 B) a similar damage at 10 µM ( p<0.001). To evaluate whether DNA strand breaks originate from alkali-labile sites or as a consequence of inhibition of topoisomerases action, studies on DNA repair were performed in MIA PaCa-2 cells. Cells were treated with the diterpene 1 or 10 , respectively, which were removed after 1h and the comet assay was performed after 12h and 24h. Treatment with sugiol ( 10 ) revealed significant recovering after 12h (about 50%) and approximately 70% after 24h, comparable to camptothecin ( p< 0.001), but at a higher concentration. Cells treated with 7 α-acetoxyroyleanone ( 1) exhibited a lower repair rate reaching only approximately 45% after 24h. Camptothecin (5 µM) was used as positive control and exhibited a 45% and 70% repair of DNA damage after 12h and 24h, respectively (see Figure 2.108). 158 Results
Alkali comet assay
10.0 *
8.0 *
A OTM 6.0 * 4.0 *
2.0
0.0 control CPT 5 µM 2 µM 4 µM 8 µM
7α-acetoxyroyleanone ( 1) 10.0
* 8.0
* B 6.0
OTM * * 4.0
2.0
0.0 control CPT 5 µM 10 µM 20 µM 40 µM
sugiol ( 10 )
Figure 2.107 - Effect of 7 ααα-acetoxyroyleanone (1) (A) and sugiol (10) (B) on DNA damage in MIA PaCa-2 cells evaluated by the alkaline comet assay after 1h treatment with the indicated concentrations. DNA damage was expressed as olive tail moment (OTM). The number of cells scored in each experiment was 100. Error bars represent the mean ± SD of two independent experiments. Significant differences between the treated groups and the control was determined with the Student’s t-test, at a level of * p < 0.001.
Results 159
1 h exposure 10.0 12 h repair
8.0 24 h repair
6.0
*
4.0 OliveTailMoment * * * * * 2.0
0.0 control CPT 5 µM (10) 40 µM (1) 4 µM
Figure 2.108 - Repair of DNA damage measured by the alkaline comet assay. MIA PaCa-2 cells were exposed for 1h with camptothecin (5 µM), sugiol [(10) 40 µM] and 7 ααα-acetoxyroyleanone [(1) 4 µM] and the DNA repair potential was evaluated after 12 and 24h. DNA damage was expressed as olive tail moment (OTM). The number of cells scored in each experiment was 100. Error bars represent the mean ± SD of three independent experiments. Significant differences were determined with the Student’s t-test, at a level of p < 0.001.
2.3.4.11 MTT assay with pan-caspase inhibitor (Q-VD-OPH)
The n-hexane extract of P. longipes and the isolated diterpenes from its extract exhibited a strong cytotoxicity proven in the MTT assay. Further results demonstrated that apoptosis does probably not play a prominent role, as caspase-3/7 activation and the effects on apoptosis- associated DNA fragmentation were only moderate. However, to have further information on the apoptotic potential 7 α-acetoxyroyleanone ( 1) and the n-hexane extract of P. longipes were exemplarily tested in the MTT assay with and without the pan-caspase inhibitor (Q-VD-OPH) using the same conditions as described in section 5.2.3.4. The results with MIA PaCa-2 cells (see Figure 2.109) showed a significant reduction in the cell death by adding Q-VD-OPH of about 58.3% and 46.1% after treatment with 1 and 2 µg/ml concentrations of the n-hexane extract of P. longipes; and about 30.2% and 37.5% after treatment with 4 and 8 µM concentrations of 7α-acetoxyroyleanone ( 1), respectively. Actinomycin D (5 µg/ml) was used as positive control and a significant ( p<0.01) reduction of about 32% was observed. From 160 Results these results can be deduced that apoptotic effects are partly, but not mainly involved in the proven cytotoxicity of diterpene 1 and the n-hexane extract of P. longipes .
MTT 100 86.3 83.9 MTT with Q-VD-OPH 80.6
75 (a) 57.8 (a) (b) 50.4 50 45.2 46.6 (b) 33.9
% cell death % cell 32.6
25 (a) 14.1
0 ActD 5µg/ml 1 µg/ml 2 µg/ml 4 µM 8 µM α extract P. longipes 7 -acetoxyroyleanone
Figure 2.109 - Determination of the apoptotic effects involved in the observed cytotoxicity of diterpene 1 (7 α-acetoxyroyleanone) and the n-hexane extract of P. longipes in MIA PaCa-2 cells after 24h exposure using the pan-caspase inhibitor (Q-VD-OPH). Error bars represent the mean ± S.E.M. of two independent experiments. Statistical analysis was calculated using the Student’s t-test at a significant level of a p < 0.01 and b p < 0.05 compared to MTT results without Q-VD- OPH.
Interestingly, the apoptotic effect is cell specific as MV-3 cells did not show any difference when using the pancaspase inhibitor Q-VD-OPH, that means the observed cytotoxicity in these cells are not due to apoptotic effects (see Figure 2.110). Results 161
75 MTT 65.4 60.7 MTT with Q-VD-OPH
50
32.8 30.8 % cell death %cell 25 20.1 21.1
6.1 6.8 4.8 4.1 0 ActD 5µg/ml 1 µg/ml 2 µg/ml 4 µM 8 µM
extract P. longipes 7 α-acetoxyroyleanone
Figure 2.110 - Determination of the proportion of apoptotic MV-3 cells within the killed cells after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7α-acetoxyroyleanone (1), studied with and without the pan-caspase inhibitor (Q-VD-OPH). Error bars represent the mean ± S.E.M. of two independent experiments.
2.3.4.12 MTT studies with the ROS-scavenging BHA
It has already been shown that abietane diterpenes with a quinone structure element can possess antioxidative activity (Wang et al. 2002; Kolak et al. 2009; Weng & Gordon 1992; Miura et al. 2002) and that ROS can be involved in apoptosis signaling (Cai et al. 2007). To study the impact of possible ROS arising by the quinone diterpenes the MTT assay was performed with and without the ROS scavenging BHA in MIA PaCa-2 cells with the n-hexane extract of P. longipes and 7 α-acetoxyroyleanone ( 1). Figure 2.111 shows that the n-hexane extract of P. longipes significantly decreased the percentage of cell death of about 22.7% and 22.3% at 1 and 2 µg/ml concentrations, respectively. Concentrations of 4 and 8 µM of 7 α- acetoxyroyleanone ( 1) also significantly reduced cytotoxicity of about 33.5% and 18.5%, respectively. Actinomycin D (5 µg/ml) was used as positive control and no differences were observed. 162 Results
100 MTT 90.2 87.8 87.5 88.0 MTT with BHA (a) (a) 75 73.4 68.4
50 46.0
(b)
% cell death %cell 33.5 (b) 30.6 25.9 25
0 ActD 5 µg/ml 1 µg/ml 2 µg/ml 4 µM 8 µM
extract P. longipes 7 α-acetoxyroyleanone
Figure 2.111 - Influence of ROS scavenger BHA on cell death in MIA PaCa-2 after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7 α- acetoxyroyleanone (1). Error bars represent the mean ± S.E.M. of two independent experiments. Statistics analysis was calculated using the Student’s t-test at a significant level of a p<0.01 and b p<0.05 compared to MTT results without BHA.
The same studies using MV-3 cells were performed and the results are shown in Figure 2.112. This time no significant influence was observed with the n-hexane extract as well as with 7 α-acetoxyroyleanone ( 1).
100 88.4 88.9 MTT
MTT with BHA 75 67.3 69.2 63.1 60.5
50 % cell death % cell
25 17.7 14.8
1.6 3.5 0 ActD 5µg/ml 1 µg/ml 2 µg/ml 4 µM 8 µM
extract P. longipes 7 α-acetoxyroyleanone
Figure 2.112 - Influence of ROS scavenger BHA on cell death of MV-3 after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7 α-acetoxyroyleanone (1). Error bars represent the mean ± S.E.M. of two independent experiments. Results 163
2.3.4.13 ROS quantification analysis
To confirm that ROS may be involved to a small part in the proven cytotoxicity of the n- hexane extract and its diterpenes we examined the impact on ROS accumulation. Besides the n-hexane extract three diterpenes ( 1, 4 and 10 ) were included in the study. To assess changes in intracellular ROS levels, we used the oxidation-sensitive fluorescent probe DCFH-DA (5,6- carboxy-2,7-dichlorofluorescein-diacetate). DCFH-DA can be taken up into the cells, and then oxidized by ROS to its fluorescent derivative DCF, which can be detected by fluorescence measurement (Schmich 2011). As shown in Figure 2.113, the n-hexane extract and the tested compounds did not induce any significant increase in intracellular ROS amounts, except of diterpene 1, which was at least moderately active. These results should be considered as preliminary, as they have not been reproduced.
2
1.6 1.6
1.5 1.4 1.3 1.2 1.2 1.2 1.2 1.2 1.1 1.0 1.0 1.0 1 0.8 fold increase fold
0.5
0 control ActD 2 µg/ml 5 µg/ml 10µg/ml 2 µM 4 µM 8 µM 20 µM 40 µM 80 µM 20 µM 40 µM 80 µM 5µg/ml extract P. longipes 7α−acetoxyroyleanone 7-ketorroyleanone sugiol
Figure 2.113 - ROS accumulation after MIA PaCa-2 cell exposed to the extract of P. longipes , 7α-acetoxyroyleanone (1), 7-ketoroyleanone (4) and sugiol (10) for 24h with the indicated concentrations. ROS was measured using the dichlorofluorescein fluorescence assay and referred to untreated control.
2.3.4.14 Inhibitory activity on p38 ααα MAP Kinase
The mitogen activated protein kinases (MAPKs) are a family of enzymes that catalyze the phosphorylation of specific serines and threonines of target substrates. Such phosphorylation 164 Results is part of the cascade that converts extracellular signals to intracellular pathways, controlling a wide range of cellular responses, such as proliferation, differentiation and apoptosis. p38 inhibitors have been used in clinical trials for inflammatory diseases and also demonstrated to be involved in the regulation of cellular processes that directly contribute to tumor suppression, including oncogene-induced senescence, replicative senescence, contact inhibition and DNA-damage response, and activation of key cell-cycle regulators p53 and p73 (Hui et al. 2007; Olson & Hallahan 2004; Han & Sun 2007). Therefore, the diterpenes were investigated for their inhibitory effects on p38 α MAPK as described in section 5.2.3.13. As observed in Table 2.22 those compounds with a para -naphthoquinone system together with a substitution at C-7, as an acetyl group (1) or keto group ( 4) were the most active with an IC 50 value about 3 µM.
Table 2.22 - Inhibitory activity of the n-hexane extract of P. longipes and the respective isolated diterpenes on p38 ααα MAPK. IC 50 values are given in µM and represent the mean ± S.E.M. for n=3. SB203580 was used as reference substance and showed an IC 50 of 0.017 µM (± 0.002).
Tested compound IC 50 (µM)
Ext. P. longipes* 1.52 ± 0.32
7α-acetoxyroyleanone ( 1) 3.47 ± 0.44
Horminone ( 2) 15.56 ± 2.09
Royleanone ( 3) 9.96 ± 0.39
7-ketoroyleanone ( 4) 3.92 ± 0.33
Iguestol ( 6) 16.32 ± 1.59
Deoxyneocryptotanshinone ( 7) 3.78 ± 0.92
12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one ( 8) 88.60 ± 3.91
Inuroyleanol ( 9) 24.61 ± 2.14
Sugiol ( 10 ) 15.70 ± 0.24
* IC 50 value in µg.
Discussion 165
3. Discussion
3.1. Wound healing effects of traditional medicinal plants using the scratch assay
Wound healing is a complex mixture of different processes including inflammation, cell proliferation and migration, and the remodeling or maturation of the new tissue. In addition, healing of the wounds is impaired by microbial infection and destruction of cells and tissues by reactive oxygen species. Since ancient times, people have used plants and preparations thereof to accelerate the wound healing process (Schmidt et al. 2009; Reuter et al. 2010). Mostly, a plant extract used for wound healing may affect more than one of these processes (Gurtner et al. 2008; Houghton et al. 2007; Singer & Clark 1999). Their use is often merely based on tradition, without any scientific evidence of efficacy and little knowledge about putative active compounds or their mode of actions . In vitro tests have been proven to be useful in bioactive-guided fractionation and determination of the active compounds in plant extracts used in traditional medicine for wound healing. Despite their widespread use, it is important to keep in mind that in vitro assays mostly study one aspect and are concentrated on one target. However, diseases including wounds are often complex processes and several mechanisms are involved. Nevertheless, they can offer valuable information and also provide insights in the mode of action of possible therapeutic compounds, but they can never replace an in vivo model which should be used as final proof for the therapeutic efficacy (Houghton et al. 2005; Potterat & Hamburger 2006). Concerning the available in vitro wound healing assays there is still a need for a suitable assay providing sufficient quantitative information on fibroblast proliferation and migration during the healing process and which can be easily performed. Therefore, in this thesis the scratch assay, which has been proven as a valuable in vitro model and used for different purposes (Liang et al. 2007; Ranzato et al. 2008; van Horssen et al. 2006) was optimized to be a suitable tool for the first evaluation on the rebuilding of new granulation tissue, and consequently for successful wound closure. 166 Discussion
In general, the scratch assay, described in this thesis, possess some advantages compared to the other available models (Gough et al. 2011; Matsubayashi et al. 2011; Yarrow et al. 2004; Zordan et al. 2011; Geback et al. 2009). The scratch assay mimics the in vivo situation as the ruptured cells release chemotactic agents, as cytokines and growth factors. This effect is not observed in the Oris ® assay or stopper-based assay for example, where a plate containing a small stopper prevents the cells from growing in the central circular section of the well. When the stopper is removed, the cells can migrate into the free area (Gough et al. 2011). The optimized scratch assay is also considered as ideal for migratory studies of adherent monolayer cell types that can not be evaluated by the trans-well migration assay or Boyden chamber assay (Boyden 1962). The Boyden chamber assay uses a two-chamber system separated by a microporous membrane. Chemotactic agents are added to the lower chamber, and the cells that move through the membrane, induced by chemokines, are counted. This method is ideal for analysis of nonadherent cells. However, adherent cells migrate through the pores and adhere to the underside of the membrane which makes quantification difficult and not representative. Moreover, the optimized assay allows differentiation between migration and proliferation effects by adding an antimitotic agent, as described by Nasca et al. 1999 and Schreier et al. 1993. Compared to the existing assays, the optimized scratch assay in this thesis also allows an accurate assessment of the percentage of cell numbers which move to the artificial wound; is cost-effective and relatively simple to set up in a cell culture laboratory. Nevertheless, as described for other assays, it still remains the difficulty to create a reproducible cell-free area with uniform dimensions. Altogether, the assay established during this thesis turned out to be a convenient and inexpensive model for the first differentiation between medicinal plants with a potential wound healing activity ( Calendula ) and those plants which have a more pronounced anti- inflammatory activity ( Matricaria ). Our results with Calendula extracts are in line with the assumption that Calendula preparations stimulate granulation (Brown & Dattner 1998; Klouchek-Popova E et al. 1982; Rao et al. 1991), as fibroblasts, which are mainly involved in granulation tissue, were stimulated by Calendula extracts at low concentrations resulting in proliferation and migration within the wound site. Moreover, these results also confirmed previous in vivo studies. Here, the efficacy of Calendula extracts has been demonstrated (Klouchek-Popova E et al. 1982; Rao et al. 1991) including also a phase III trial in the prevention of acute dermatitis during irradiation for breast cancer (Pommier et al. 2004). However, despite of further trials summarized in Leach 2008 the use of Calendula containing Discussion 167 herbal products is mainly based on folk medicine, and detailed mechanistic studies are missing (Leach 2008). The effects of Calendula in increasing the cell number of fibroblasts in the scratched area were due to the migration of cells and proliferation of the migrating cells. By addition of mitomycin C, which blocks mitosis and thus allows to discriminate between stimulation of migration and proliferation (Schreier et al. 1993), the cell number decreased to about 25% in the artificial wound after Calendula and platelet-derived growth factor (PDGF) treatment, respectively. Interestingly, the result with PDGF does not agree with studies in literature where PDGF exclusively is reported to stimulate migration (Schreier et al. 1993). However, as the proliferation effect is not very pronounced, this discrepancy may be explained by differences in the experimental conditions. Triterpenoids, in particular faradiol monoesters, are considered as the active components of the anti-inflammatory activity of Calendula (Dellaloggia et al. 1994). The compounds which are responsible for the wound healing effects are still unknown (Matysik et al. 2005). Some compounds, as the carotenoids, have been discussed to affect the function of fibroblasts, but this could not experimentally be proven (Schneider E. et al. 1991). Here, it could be demonstrated that triterpene monoesters, e.g. faradiol palmitate and faradiol myristate, from Calendula stimulate fibroblast proliferation and migration. Both faradiol esters showed a comparable significant increase in the cell number in the scratched area. Further studies revealed a similar decrease (about 30%) in cell numbers when the cells were pretreated with the proliferation inhibitor mitomycin. This effect was also shown for the n-hexane extract of Calendula . Taken together, these results show that triterpenes may positively influence the wound healing effect of Calendula extract by stimulating the proliferation and, to a higher extent, the migration of fibroblasts. The n-hexane extract showed a higher activity in the scratch assay than the isolated triterpenes when both were tested at 10 µg/ml. This indicates that the triterpenes contribute to the wound healing effect of Calendula extracts. However, further compounds likely contribute to the wound healing activity of Calendula flowers. Altogether, our studies extended the current knowledge on the wound-healing effects of the n- hexane and ethanolic extract from Calendula officinalis .
168 Discussion
3.2. Peltodon longipes
The discovery of biologically active natural products has always been a long and tedious process requiring substantial amount of material. The development of hyphenated techniques introduced a tremendous advance in this process and becomes an important tool for the isolation of single compounds from complex mixtures containing many constituents in widely different concentrations and possessing a broad spectrum of physical and chemical properties (Potterat & Hamburger 2008; Verpoorte 2000; Jaroszewski 2005b; Jaroszewski 2005a). Here, intensive phytochemical and biological studies were performed with Peltodon longipes to find out the effective compounds. This plant was selected from a screening study with 40 plant extracts belonging to different families. All plants have been used in traditional medicine in south of Brazil due to their antimicrobial, cytotoxic and anti-inflammatory properties (Schmidt 2011; Schmidt et al. 2009). During the screening phase, the n-hexane extract of Peltodon longipes showed a high cytotoxicity, caspase-3 activation and antibacterial activity. P. longipes is locally known as “baicuru amarelo” or “hortela do mato” and has been externally used in folk medicine mainly as an anti-inflammatory and antiseptic remedy (Mentz et al. 1997). No phytochemical studies have been described in the literature. The genus Peltodon belongs to the Lamiaceae family and comprises 8 species (Harley et al. 2004). So far, in the genus Peltodon only the species P. radicans has been studied and reported to possess antiedematogenic activity against the venom of Bothrops atrox due to the presence of aliphatic hydrocarbons (da Costa et al. 2008). In the beginning, the presence of abietane diterpenes was demonstrated by LC-NMR in the n-hexane extract of the roots of Peltodon longipes . Therefore, one of the aims of this thesis was to do the isolation and structure elucidation of these compounds. Abietane diterpenes have hydrophilic properties and many papers describe their isolation using silica gel as stationary phase and elution with n-hexane-EtOAc (Delatorre et al. 1992; Fraga et al. 2005; Maldonado et al. 1994; Nagy et al. 1999b; Nagy et al. 1999a) or petrol-EtOAc (Galicia et al. 1988; Rodriguezhahn et al. 1989; Ulubelen et al. 1987; Ulubelen et al. 1995). Hence, fractionation of the n-hexane extract of Peltodon longipes was performed by open column and low pressure liquid chromatography using silica gel and afforded 12 abietane diterpenes. The isolated diterpenes were identified on the basis of their mass spectra and 1D ( 1H and 13 C) and 2D NMR (COSY, HSQC and HMBC) data as the known 7α-acetoxyroyleanone (1), 7α-hydroxyroyleanone (syn. horminone) (2), royleanone (3), 7-ketoroyleanone (4), 7α- Discussion 169 ethoxyroyleanone (5), iguestol (6), deoxyneocryptotanshinone (7), 12-hydroxy-11- metoxyabieta-8,11,13-trien-7-one (8), inuroyleanol (9), sugiol (10 ), cryptojaponol (11 ), and orthosiphonol ( 12 ). All MS data and NMR data agreed with those reported in the literature (for structures see Figure 2.93).
3.3. Biological studies of abietane diterpenes
3.3.1. Cytotoxic studies of abietanes from Peltodon and Salvia Species Many different biological activities and targets have been addressed for the abietane diterpenes such as antibacterial (Lee et al. 1999b; Ulubelen et al. 2001), antileishmanial (Sairafianpour et al. 2001; Tan et al. 2002), antifungal (Kusumoto et al. 2010), anti- inflammatory (Chao et al. 2005), trypanocidal (Herrera et al. 2008), antioxidant (Miura et al. 2002), cardiovascular (Cheng 2007) and cytotoxic (Araujo et al. 2006; Jonathan et al. 1989; Moujir et al. 1996; Slamenova et al. 2004; Marques et al. 2002) among many others. We have focused our biological studies on their cytotoxic activity and the respective molecular mechanism of action. All isolated compounds belong to the abietane-type diterpenes which are characteristic secondary metabolites of the Lamiaceae family (Ikeshiro et al. 1991; Rüedi 1984; Sairafianpour et al. 2001; Topcu & Goren 2007). Among this class of diterpenes tanshinones deserve special interest due to presence of an ortho - or para -quinone or lactone ring C, and a furan or dihydrofuran ring D. Tanshinones have been repeatedly isolated from different Salvia species (Chang et al. 1990; Fraga et al. 2005; Ryu et al. 1997; Spiridonov et al. 2003; Tezuka et al. 1998; Ulubelen et al. 1999b). The abietanes from P. longipes (1 - 12 ) as well as eight diterpenes from Salvia miltiorrhiza (13 - 19 ) (Slusarczyk et al. 2011), and two from Salvia sahendica (20 and 21 ) (Jassbi et al. 2006b) were investigated for their cytotoxic activities and first structure activity relationships were drawn. Interestingly, the para -naphthoquinone skeleton was not a prerequisite for cytotoxic effects, as discussed by Sairafianpour et al. 2001, Topcu & Goren 2007 and Wang et al. 2007. Whereas diterpenes 1 - 5 possessed a para -naphthoquinone moiety and exhibited a pronounced cytotoxic effect, the para -quinone abietane 7 was only weakly cytotoxic and the non-quinoidal compounds 6, 8 and 10 were moderately active. Considering only the abietane diterpenes with an ortho -quinone moiety our results confirm the recent structure activity relationship proposal of Wang et al. 2007 who suggested an 170 Discussion ortho -quinone moiety in ring C, an intact ring D, and a relatively planar structure as relevant structural features for cytotoxicity of these types of diterpenes. However, as the para -quinone derivative 1 also exhibited a strong cytotoxic activity, an ortho -quinone in ring C and the presence of ring D seems to be not the only structural requirements for a high cytotoxicity. Compound 1 was already shown by Araujo et al. 2006 to possess a high cytotoxic effect in five other cancer cell lines. Notably, despite the fact that ortho -quinones were generally more active than the para -quinones, activities of the crude extracts of P. longipes with the dominance in para -quinone diterpenes and S. miltiorrhiza in which abietane form the ortho - quinone type occur were comparable (IC 50 1.3 and 1.8 µg/ml, respectively). Summarizing our cytotoxic studies with 21 abietane and one secoabietane derivative, no straightforward structural prerequisites for strong cytotoxic activity could be described. However, one may have to take into account that the underlying mechanisms for cytotoxicity of the investigated diterpenes may differ. The cytotoxic studies revealed that the diterpenes are probably the main bioactive compounds in the roots of P. longipes and responsible for the observed cytotoxic effects. Moreover, studies with 7 α-acetoxyroyleanone ( 1) which were carried out by the NCI showed remarkable differences in specificity depending on the cell line studied. Thus, an LC 50 of about 7.4 µM was observed in nine melanoma cell lines, whereas in six different Leukemia cells the LC 50 could not be determined (>100 µM). Further studies may explain this interesting observation.
3.3.2. Studies on the alkylating properties of diterpenes from Peltodon longipes The presence of a quinone group in the structure of a compound is often correlated with significant increased cell death by a mechanism that involves DNA damage by single and double-strand breaks, free radicals and reactive oxygen species generation (Begleiter & Blair 1984). These activities are enhanced when the quinone agent had the ability to bind to the DNA by alkylation (Begleiter 1985). As some of the isolated diterpenes from P. longipes also possess a quinone structural element the question arises whether their observed cytotoxic effects may be due to the presence of these highly reactive functional groups which can serve as Michael acceptors and function as alkylation agents. It is known that such quinones can react with cellular nucleophiles ( e.g. cysteinyl thiols) or crucial cellular proteins forming Discussion 171 covalently linked Michael adducts that induce cell damage (Katritzky et al. 2008; Bolton et al. 2000; Bolton et al. 1997; Begleiter 1985). Alkylation activity was proven for diterpenes 1 - 3 in an extracellular system using the nucleophile 4-(4-nitrobenzyl) pyridine (NBP) as the alkylating substrate. NBP is considered as an effective trap for alkylating agents with nucleophilic characteristics similar to DNA bases and an important tool for preliminary information about the reactivity of such compounds (Kim & Thomas 1992; Thomas et al. 1992). Diterpenes 1 - 3 all possess a para - quinone structure and react in a similar way with the nucleophile NBP, but exert different cytotoxic effects. Therefore, it can be concluded that the cytotoxic effects of these compounds can only partially be explained by their alkylating properties and that further mechanisms should be involved. Notably, the reactivity observed for compound 1 was significant only at higher concentrations (> 10 µM) and not at the concentrations used in the biological studies (0.5 - 8 µM). Moreover, diterpenes 4 and 10 which display similar or even higher cytotoxicity compared to 2 and 3 did not react with NBP. Alkylating agents have been used in cancer therapy for many decades, and their number is still growing, especially in those cancers with high proliferating rates. Several important clinical drugs ( e.g . cisplatin, and the natural product mitomycin C) are known to induce DNA interstrand cross-links causing subsequent DNA strand breaks which disturb cell cycle progression and trigger cell death through apoptosis (Wang et al. 2005; Lee et al. 2010a). Altogether, alkylating agents are considered as double edged swords that are capable of great destruction and even greater healing (Ralhan & Kaur 2007; Hurley 2002).
3.3.3. Diterpenes from Peltodon longipes and their effects on topoisomerases Diterpene-mediated cell death was further characterized by studying their effects on topoisomerases. Previously it was shown that 1,2-dihydrotanshinone ( 16 ), cryptotanshinone (14 ) and 7-ketoroyleanone ( 4) inhibit topoisomerase I (Han et al. 2008; Lee et al. 1999a). Currently, there are two major human DNA topoisomerases, topoisomerase I (topo I) and topoisomerase II (topo II) that have been tightly established to be effective molecular targets for many antitumor drugs (Nitiss 2009; Pommier 2006; Pommier 2009). The Eukaryotic DNA topo I is an enzyme that acts to relax supercoils generated during transcription and DNA replication. Topo I mediates DNA relaxation by creating a temporary 172 Discussion single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723) at a DNA phosphodiester bond forming a 3 ′-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5 ′-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3 ′-phosphotyrosyl topo I-DNA complex remains low (Champoux 2001; Li & Liu 2001). Drugs targeting topo I can be divided into two broad classes. The topo I interfacial inhibitors that stabilize the covalent enzyme-DNA complex specifically by blocking the DNA religation (Pommier & Cherfils 2005; Pommier 2009) and the catalytic inhibitors which usually bind to the topo I as well as to the DNA and block the interaction between the enzyme and DNA or inactivate the complex (Wu et al. 2010; Zhai et al. 2008; Pommier 2009). In contrast to topo I, type II topoisomerase acts by passing an intact DNA double helix through another double helix that has been cleaved by the enzyme, requiring a complex conformational change in the enzyme that is fueled by ATP hydrolysis. Following DNA strand passage topoisomerase II religates the cleaved strand. Eukaryotic cells encode two isoforms of topoisomerase II, α and β, which perform functions encompassing replication, transcription and DNA repair. Topoisomerase II α has been studied most extensively. This isoform is associated with replication and is essential for chromosomal segregation. Consistent with these functions its expression peaks appear at G2/M phase of the cell cycle (Wang 2002; Champoux 2001). Drugs targeting topo II are mainly divided into two broad classes. The first class includes most of the clinically active agents, such as etoposide, the anthracycline doxorubicin and mitoxantrone (Nitiss 2009). They act by increasing the levels of topo-II-DNA covalent complexes converting the transient DNA double strand breaks into permanent lesions. They have been named as topo II poisons. Topo II poisoning may be due to direct interaction of the drug with the enzyme, or by alterations in DNA structure inhibiting the religation. The second class of topo II inhibitors is thought to kill cells through the elimination of the essential enzymatic activity of topo II and is therefore described as topo II catalytic inhibitors (Salerno et al. 2010; Nitiss 2009). The natural alkaloid camptothecin (CPT) is a classical example of a topo I poison. Topo I is the unique intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S- Discussion 173 phase specific (Pommier 2006; Pommier 2009; Darzynkiewicz et al. 1996). When the DNA replication fork and/or transcription machinery collide with such complex, single strand breaks are converted in double strand breaks. This processes activate different signaling pathways driving the cells towards DNA damage repair, or when the damage is too severe to ultimate cell death, apoptotic cell death or other modality such as senescence (Beretta et al. 2008; Pisano et al. 2008). CPT acts as an uncompetitive inhibitor and specifically targets the enzyme-substrate complex. CPT binds at the site of the DNA cleavage by intercalating between the DNA base pairs and mimics a DNA base pair thus inhibiting the religation of the cleaved strand (Li & Liu 2001; Liu et al. 2000; Staker et al. 2002; Pommier et al. 1998). The stacking model has been validated by crystal structures of the topo I - DNA - CPT ternary complex (Staker et al. 2002; Staker et al. 2005). Similar mode of action has also been proven for non-CPT inhibitors, such as indenisoquinolines (Pommier & Cherfils 2005; Pommier 2009). Although most of the studied natural and/or synthetic compounds have been demonstrated to stabilize the cleavage complex, some examples are found in the literature for a direct interaction with the topoisomerase I as well as with the DNA resulting in inhibitory effects. An example for this kind of interaction is the flavone luteolin (Chowdhury et al. 2002). Moreover, the ternary complex formation can also be inactivated (Pommier et al. 1998). β - lapachone, a member of the ortho -naphthoquinone family is an example for a direct interaction with topoisomerase I rather than with the DNA (Li et al. 1993). Additionally, a further possibility to inhibit the function of topo I was shown with an indolizinoquinoline- 5,12-dione derivative (CY13II). It was proposed that this compound binds to topo I without affecting the binding of the enzyme to the DNA, but the drug-enzyme-DNA ternary noncovalent complex is catalytically inactive and prevents the cleavage activity of topo I (see also Figure 3.1 D) (Wu et al. 2010). Altogether, many natural and synthetic compounds have been reported to exhibit the ability to interfere with topoisomerase I (Salerno et al. 2010; Xia et al. 1999). Their discussed mode of actions are summarized in Figure 3.1 (Webb & Ebeler 2003; Wu et al. 2010). 174 Discussion
topo I topo I-DNA topo I-DNA cleavage controlled complex complex DNA rotation
binding religation A cleavage release
CPT
drug B C +
drug D
Figure 3.1 - A - Proposed mechanism by which topoisomerase I controls the topological state of DNA at normal conditions. B - inhibitors of the topoisomerase-DNA cleavage complex, “interfacial inhibitors”. C and D - topoisomerase I catalytic inhibitors (Adapted from Wu et al. 2010). (A) - (1 st ) topo I binds to supercoiled DNA substrate to form the noncovalent topo I - DNA complex; (2 nd ) topo I catalyzes the cleavage of one DNA strand to form a transient topoisomerase cleavage complex; (3 rd ) controlled rotation and release of the supercolied tension of DNA; (4 th ) the cleaved DNA strand is religated; (5 th ) topo I is released from the relaxed DNA and undergoes another cycle of DNA relaxation (Pommier 2006). ( B) - Classical topo I poison, camptothecin (CPT), intercalates between the DNA base pairs at the topoisomerase cleavage site and stabilizes the topoisomerase cleavage complexes. The intercalation inhibits the religation of DNA and the release of topo I (Pommier 2006; Pommier 2009). ( C) - Catalytic inhibitor binds to topo I as well as to the DNA and subsequently blocks the interaction between topo I and DNA (Shuai et al. 2009; Zhai et al. 2008; Boege et al. 1996). When these drugs bind to the enzyme or the DNA, they prevent all subsequent steps of the catalytic cycle. ( D) - The catalytic inhibitor which binds to topo I as well as to the DNA, allows the complex formation (drug-enzyme-DNA) but prevents the catalytic activity of topo I (Boege et al. 1996; Wu et al. 2010; Chowdhury et al. 2002; Frydman et al. 1997; Li et al. 1993).
Considering the different mode of actions the question arises how the inhibitory activity of the diterpenes from Peltodon longipes can be explained. The diterpenes 1 - 4 and 10 significantly inhibited human topoisomerase I relaxation activity at low concentrations
(results details in section 2.3.4.3). Diterpenes 4 and 10 were the most active ones with IC 50 values of 2.8 and 4.7 µM, respectively. CPT exhibited an IC 50 of 28.0 µM. Both diterpenes also showed significant inhibitory activity against topoisomerase II relaxation activity, but with higher IC 50 values of 26.7 µM and 26.0 µM, respectively. Etoposide gave an IC 50 of 58.2 µM. Hence, the diterpenes preferentially inhibit topoisomerase I relaxation activity. Diterpenes were added to the reaction mixture before the enzyme. Therefore, it can be assumed that the diterpenes either bind to the DNA prior to the enzyme or to the enzyme thus inhibiting or inactivating the complex formation and consequently the relaxation of supercoiled DNA (Webb & Ebeler 2003). Discussion 175
As diterpenes 1 - 3 exhibited alkylating properties in the extracellular system using the nucleophile NBP, they may probably directly target the DNA in a nonspecific way and thus interfering with topoisomerase activity. A similar mechanism is discussed for the topo II inhibitory activity of the diterpene clerocidin which alkylates the nitrogen on position 7 of guanine residues and which nick supercoiled DNA (Gatto et al. 2001). Diterpenes 4 and 10 did not exhibit alkylating properties and only a moderate cytotoxicity
(IC 50 of 30.1 and 17.9 µM, respectively) in MIA PaCa-2 cells. However, they showed higher inhibition of topoisomerase I and II relaxation activity compared to compounds 1 - 3 and to the respective positive controls camptothecin and etoposide. Based on these effects together with the non-planar stereochemical conformation, that hinders the intercalation with the DNA, it can be supposed that these diterpenes may act in a different way towards topo I as CPT and the diterpenes 1 - 3 (Pommier & Cherfils 2005; Pommier 2009). They may directly target topo I as discussed for the ortho -quinone derivative β-lapachone. For this natural compound a direct interaction with topo I is suggested that blocks the formation of cleavable complex, although the affected enzyme still binds to DNA substrate (Frydman et al. 1997; Li et al. 1993). Moreover, inhibition of topo I was also shown for dihydrotanshinone I, an abietane with an ortho -quinone group. It was discussed that this diterpene inhibits the catalytic activity of topo I by the formation of a cleavable complex resulting in DNA damage (Lee et al. 1999a). In this thesis it was shown that the diterpenes 4 and 10 inhibited both topoisomerases, although with a prevalence for topo I. This property may be beneficial in cancer therapy. Therefore, one approach in cancer research is the development of dual topo I and II inhibitors as anticancer drugs. These drugs may have the advantage of reduced toxic side effects. Moreover, dual topo I and II inhibitors may also avoid failure of clinical therapies due to resistence of topo I inhibitors which is often accompanied by a concomitant rise in topo II levels (Salerno et al. 2010). Compounds acting as dual topo I and II inhibitors have already been described. Interestingly, some of them, such as the triptycene bisquinones also possess a para -quinone structural element. A possibility to explain in silico the mechanism of the observed inhibition of DNA relaxation is by molecular modeling which can also be a helpful tool to predict a molecular target or to predict a possible activity towards a specific target (Lauria et al. 2007). Our docking studies using the co-crystallized structure of topoisomerase I, DNA and camptothecin (Brenke et al. 2009) revealed that the mode of action of the diterpenes differs from that of 176 Discussion
CPT since no correlation between experimental data and the docking scores were observed when CPT and the diterpenes were docked in the characteristic binding pocket known for CPT (Fan et al. 1998). The non-planar conformation of the diterpenes makes an intercalation difficult within the cleaved site of the DNA as observed for CPT. However, molecular docking provided evidence that these diterpenes act as noncamptothecin inhibitors and show an alternative binding to topo I. According to molecular docking experiments diterpenes 1 - 4 and 10 do not form a drug-enzyme-DNA covalent ternary complex as observed for CPT, but may directly interact with topo I. A direct targeting of the DNA and consequently inhibition of the catalytic activity of topo I is unlikely. The predicted binding pocket may be used to design more effective topo I inhibitors starting from diterpene 4 or 10 as leads. This approach has already been successfully done with betulinic acid (Bar et al. 2009). Inhibition of topoisomerases is most often accompanied by DNA strand breaks. The classical approach for detecting topoisomerase-induced DNA strand breaks is the comet assay, a useful technique which allows the assessment of low levels of DNA strand breaks in single cells (Collins 2004; Olive & Banath 2006). Therefore, the comet assay was performed to study the respond of the cells to topoisomerase-mediated DNA damage. MIA PaCa-2 cells exposed to diterpene 1 and 10 exhibited DNA tailing at different levels, indicating an increased electrophoretic mobility of the DNA fragments from the nuclear DNA and confirming the presence of strand breaks. In contrast, the nuclei of control cells presented a compact round area of fluorescence and only a slight DNA tail was detected. Notably, diterpene 1 which was ~2 times more effective in the topo I relaxation assay (IC 50 14.2 µM) than CPT (IC 50 28 µM) exhibited not so much DNA damage compared to CPT. The amount of DNA strand breaks induced by compound 1 at a concentration of 8 µM (6.6 OTM) after 1h exposure was smaller than that of CPT at a 5 µM concentration (8.2 OTM). CPT also was ~10 times more cytotoxic than compound 1 in MIA PaCa-2 cells (IC 50s 0.4 and 4.7 µM, respectively). When cells were transferred to drug-free medium and allowed to recover for 12 and 24h, once more, significant differences were observed between CPT and diterpene 1. While the remaining level of DNA damage with CPT-treated cells after 24h in a drug-free medium was less than 30% diterpene 1-treated cells still display ~55% of persistent DNA damage. These results suggest that the cytotoxic effects and the DNA damaging capacity of diterpene 1 may not be exclusively related to topoisomerase inhibition, but that further factors such as the ability to directly interact with the DNA and form covalently linked Michael adducts (an irreversible form of DNA damage) should also be considered. Consisting with our Discussion 177 results Slamenova et al. 2004 has also reported cytotoxic effects and DNA-strand breaks using the comet assay of diterpene 1. They observed that diterpene 1 induced DNA damage at relatively low concentrations in Caco-2 cells at pH > 12.5. The authors suggested that these effects were not due to an enzymatic (endonuclease or topoisomerase) action but originated from alkali-labile DNA adducts. The kinetics of strand DNA breaks rejoining was also slow and comparable to MNNG, a strong alkylating agent. Compound 10 displays even more pronounced differences between the topoisomerase inhibitory activity and the ability to induce DNA damage. A ~6 times higher ability to inhibit the relaxation of pBR322 DNA (IC 50 4.7 µM) than CPT (IC 50 28 µM) was observed for diterpene 10 . However, this effect on topoisomerase seems not be converted in DNA strand breaks, as even at higher concentrations (40 µM = 5.8 OTM) diterpene 10 did not exhibit the same effects as CPT (5 µM = 8.2 OTM). Interestingly, diterpene 10 was able to rejoin DNA strand breaks after 24h to approximately 70%, the same rate as obtained for CPT. Differences between topoisomerase activity, cytotoxicity, and ability to induce DNA strand breaks have also been reported in literature. The novel fluorinated lipophilic epipodophylloid (F11782), a dual catalytic inhibitor of topoisomerase I and II, exhibited some similarities with our results. F11782 inhibited pBR322 relaxation induced by topoisomerase I with an IC 50 value of 4.2 µM (Perrin et al. 2000). A comparison between DNA damage induced by F11782 with that of CPT revealed that F11782 initially induced less DNA strand breaks than CPT. However, a time-course experiment revealed that CPT reached the maximum of DNA damage after 1h and then a plateau. F11782 could induce the same DNA damage level after 4h and this damage increased with the time. These results suggest that the amount of DNA-strand breaks induced by F11782 accumulate within the exposition time and was not only dependent on the concentration of F11782, and that it may be a cumulative cellular process (Barret et al. 2000). The biflavonoid genistein exhibited similar properties to induce topoisomerase II-DNA cleavage complex compared to etoposide. However their effects on cell death and capacity to generate permanent DNA strand breaks were markedly different. Genistein only displayed weak cytotoxic activity in CEM cells with an IC 50 of ~180 µM, while etoposide exhibited an
IC 50 of ~10 µM. Induction of histone H2AX phosphorylation, a suitable biomarker for DNA double strand breaks, showed that etoposide was ~10 times more efficient in converting the enzyme-DNA intermediates in permanent DNA damage than genistein. Etoposide-induced cleavage complexes were also ~10 times more stable than the genistein-induced complexes (Bandele & Osheroff 2008). 178 Discussion
3.3.4. Diterpenes from Peltodon and their effects on cell cycle Inhibition of topoisomerases and subsequent DNA strand breaks can influence the cell division and cell cycle progression. Therefore, the effect of diterpenes 1 - 4 and 10 on the cell cycle was analyzed. Diterpene 1 exhibited a significant decrease in the cell number at the G0/G1 phase and arrested the cells in the S phase at low concentrations (1 µM) thus inhibiting the cells to enter mitosis. In contrast, diterpenes 4 and 10 led to an accumulation of cells in the G0/G1 phase and reduced cell number in the S phase, indicating that cells continue mitosis but can not pass the G1 phase to enter the S phase and fail to trigger DNA synthesis. However, higher concentrations were needed to significantly inhibit the cell cycle progression (compound 4 - 30 µM and 10 - 20 µM). Compounds 2 and 3 did not significantly influence the cell cycle up to a tested concentration of 40 µM. The concentration necessary to arrest the cell cycle was comparable with that one necessary to kill the cells in the MTT and to induce DNA strand breaks, but not to inhibit topo I. Therefore, it can be assumed that topoisomerase may not be the preferential target responsible for the observed cell death and that yet unknown proteins may be influenced by the diterpenes. Progression of the cell cycle is mainly controlled by a series of cyclin-dependent kinases (CDKs). The activity of these enzymes is regulated by several mechanisms, including association with regulatory subunits (cyclins), phosphorylation and dephosphorylation, and interaction with CDK inhibitors. Because of their critical role in cell cycle progression and cellular transcription, as well as the association of their activities with apoptotic pathways, the CDKs comprise attractive set of targets for novel anticancer drug development (Shapiro 2006; Deep & Agarwal 2008). Accordingly, diterpenes 1 and 10 were tested for their ability to inhibit the main CDKs- cyclin complexes directly involved in driving the cell cycle, namely CDK1/CycB1, CDK2/CycE, CDK3/CycE, CDK4/CycD3, CDK6/CycD1, CDK7/CycH/MAT1, CDK8/CycC, CDK9/CycT and PCTAIRE1. None of the effective CDKs associated with their respective cyclin subunit was inhibited at a significant level. These results suggested that another factors may control the DNA damage checkpoint, such as the upstream kinases ATM and ATR or CDK inhibitors (Bartek et al. 2004). Arrest in the G1 and S phase induced by diterpenes 10 and 1 activates checkpoints that prevent the replication of damaged DNA. The S-phase checkpoint activated after DNA Discussion 179 damage is mainly mediated by two parallel pathways involving the upstream signaling kinases ATM and ATR and results in a rapid and transient inhibition of DNA synthesis. The first of these pathways requires the activation of cyclin 1 and cyclin 2 kinases, both of which target cdc25A phosphatase for degradation resulting in an impairment of CDK2 activation. The second pathway involves the MRN complex, MRE11-NBS1-RAD50. The MRN complex is of structural and functional importance during the early DNA double strand breaks response, by connecting DNA ends and recruiting ATM, thereby generating signaling complexes comprising damaged DNA and catalytic active ATM (Reinhardt & Yaffe 2009; Riches et al. 2008). Resveratrol is an example that inhibits topoisomerase II catalytic activity causing DNA-strand breaks. One plausible mechanism responsible for the chemopreventive activity of resveratrol in ovarian cancer cells may occur through ATM/ATR - dependent DNA damage and S-phase arrest. The ATM/ATR activation induces phosphorylation of cyclin 1 and cyclin 2, followed by that of cdc25C and cdc2, and cyclin B1 accumulation (Tyagi et al. 2005).
3.3.5. Diterpenes possess further biological activities Cells respond to DNA damage or other defects by activating checkpoints that arrest the cell cycle for efficient repair. Alternatively, when the repair system fails damaged cells can undergo cell death by different processes such as apoptosis, necrosis or senescence. Apoptotic program is a latent form in virtually all cell types and occurs in response to many cellular signals including DNA damage. Many diterpenes have been shown to trigger apoptosis leading to cell death (Cai et al. 2007; de Jesus et al. 2008; Miao et al. 2003; Nizamutdinova et al. 2008b; Su et al. 2008). Diterpenes 1 - 4 and 10 only induced apoptosis in MIA PaCa-2 cells very moderately, despite their high cytotoxicity. Diterpene 1 exhibited the highest apoptotic effects. This diterpene activated caspase 3/7 in a similar range as actinomycin D. However, measurement of apoptotic cells by FACS during the cell cycle analysis only gave a rate of approximately 23% apoptotic cells. These results are in line with previous studies that also reported high cytotoxic effects in different cell lines, which were not due to induction of apoptosis (Araujo et al. 2006; Slamenova et al. 2004). Resisting to cell death, mainly by evading apoptosis, has been considered as an important property of cancer cells and a hallmark of cancer. Cancer cells have been shown to evade cell death via dysfunctional sensors of signals or errors within the apoptotic pathway itself (Hanahan & Weinberg 2000; Hanahan & Weinberg 2011). Although resistance to apoptosis is 180 Discussion closely linked to tumorigenesis, tumor cells can still be induced to die by non-apoptotic mechanisms, such as necrosis, senescence, autophagy and mitotic catastrophe (Okada & Mak 2004). Apoptosis as well as senescence is closely associated with increased intracellular reactive oxygen species (ROS) (Ewald et al. 2010). Moreover, ROS have been shown to be involved in many cellular pathways, cell growth regulation and energy production among other functions. Thus, ROS appear to be responsible for mitochondrial events, as cytochrome c release, that leads to activation of the caspase cascade (Simon et al. 2000). ROS are toxic for the cells at certain levels, due to the oxidative stress they exert by their reaction with proteins, lipids, and nucleic acids. Consequently, the correct cellular response to ROS production is critical in order to prevent oxidative damage and to maintain cell survival. In the case of cellular damage which cannot be repaired anymore, a multicellular organism is able to remove the cell for the benefit of the surrounding cells. ROS can therefore trigger both apoptotic and necrotic cell death depending on the severity of the oxidative stress (Morgan & Liu 2011). A lot of natural compounds have been reported for their antioxidative activity (Kabouche et al. 2007; Miura et al. 2002) which can be beneficial by overwhelming ROS production. In this respect quinones from Salvia miltiorrhiza have also been demonstrated to act as antioxidants. However, diterpenes 1, 4 and 10 , which were exemplarily studied for their ROS productive activity, did not elevate ROS levels. In agreement, ROS were also not significantly involved in cell death shown for diterpene 1. Interestingly, it has been reported in the literature that diterpene 10 and some other closely related compounds, such as ferruginol, exhibited significant inhibitory activity against DPPH radical scavenging (Kolak et al. 2009; Wang et al. 2002). However, it has to be considered that this test is only of preliminary relevance. A further kinase involved in a variety of cellular processes such as proliferation, differentiation and apoptosis is p38 α which belongs to the family of mitogen-activated protein kinases (MAPKs) (Hallahan et al. 2003; Holmes et al. 2003). Studies have revealed that retinoids, cisplatin and other chemotherapeutic agents activate p38 α resulting in cancer cell apoptosis (Han & Sun 2007; Reinhardt & Yaffe 2009). Interestingly, the studied diterpenes 1 -
4 and 6 - 10 displayed high inhibitory activity towards p38 α with IC 50 values lower than 24 µM. Only diterpene 8 was weakly active. Further investigations may clarify the importance of p38 α inhibition by the diterpenes on cellular processes. Discussion 181
Altogether, the phytochemical and biological studies provided evidence that the diterpenes from the abietane type are the effective compounds in Peltodon longipes roots which possess a high cytotoxic activity. Furthermore, the studies showed that these diterpenes can target several proteins or cellular constituents depending on the respective structure, such as the topoisomerase or the DNA. However, there are some questions open to completely explain the cytotoxic mode of action which should be focused on in the future.
182 Summary
4. Summary
Medicinal plants have a long-lasting tradition in the treatment of various diseases. Often they are also used for treating wounds. To have an easy to be performed, convenient and inexpensive method for the first evaluation of the wound healing potential a scratch assay was optimized. The assay provides quantitative information on fibroblast proliferation and migration in an artificial wounded area. As a proof of concept different Brazilian and European medicinal plant extracts, including the well-known Calendula officinalis, were screened for their wound healing properties. Peltodon longipes (Lamiaceae) is a native shrub found only in South America. In Brazil it is locally known as “baicuru amarelo” or “hortela-do-mato” and externally used in folk medicine as an antiseptic and anti-inflammatory remedy. Here, Peltodon longipes was for the first time phytochemically investigated. The n-hexane extract prepared from its roots afforded twelve abietane diterpenes. Structure elucidation was based on spectroscopic methods, such as 1D and 2D NMR data ( 1H and 13 C, COSY, HSQC and HMBC) and mass spectrometric analysis. The diterpenes were identified as 7α-acetoxyroyleanone (1), 7 α-hydroxyroyleanone (syn. horminone) (2), royleanone (3), 7-ketoroyleanone (4), 7α-ethoxyroyleanone (5), iguestol (6), deoxyneocryptotanshinone (7), 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8), inuroyleanol (9), sugiol (10 ), cryptojaponol (11 ) and orthosiphonol ( 12 ). HPLC analysis of the n-hexane extract was performed and all isolated diterpenes were assigned to their respective peak. Quantification studies revealed that 7 α-acetoxyroyleanone ( 1) was the main diterpene in the n-hexane extract with a concentration of 23.4%. The n-hexane extract as well as the isolated compounds from Peltodon were studied for their cytotoxic effects and the mode of action. The abietanes from P. longipes ( 1 - 12 ) as well as eight diterpenes from Salvia miltiorrhiza (13 - 19 ) and two from Salvia sahendica (20 and 21 ) were investigated for their cytotoxic effects in human pancreatic (MIA PaCa-2) and melanoma (MV-3) tumor cell lines using the MTT assay. Tanshinone IIa ( 13 ), 7 α-acetoxyroyleanone ( 1), 1,2-dihydrotanshinone ( 16 ) and cryptotanshinone ( 14 ) showed the highest cytotoxic effects in MIA PaCa-2, displaying IC 50 values of 1.9, 4.7, 5.6 and 5.8 µM, respectively. Structure-activity relationships of abietane diterpenoid quinones are discussed. Moreover, diterpene 1 was screened in the NCI 60 cell line panel and exhibited some specificity depending on the cell line. Summary 183
Using an extracellular system with the nucleophile 4-(4-nitrobenzyl)pyridine (NBP) as alkylating substrate, diterpenes 1 - 3, but not 4 and 10, were shown to exhibit alkylating properties which may play a role in their cytotoxic activities. It can be discussed that diterpenes 1 - 3 may directly interact with the DNA. Diterpenes 1 - 4 and 10 significantly inhibited topoisomerase I relaxation activity displaying IC 50 values at lower concentrations compared to camptothecin (28 µM).
Compounds 4 and 10 were the most active ones with IC 50 of 2.8 and 4.7 M, respectively.
These two diterpenes were also active towards topoisomerase II with IC 50 of 26.7 µM and
26.0 µM, respectively and hence more active than etoposide which had an IC 50 of 58.2 µM. As these diterpenes do not have any alkylating activity, both may be used as leads for the development of more potent topoisomerase inhibitors. Molecular docking studies suggested a possible interaction of the diterpenes with topoisomerase I different from that known for CPT. Docking studies revealed that the diterpenes do not form the drug-enzyme-DNA covalent ternary complex as observed for CPT. A new binding mode was modeled, suggesting a direct interaction of the diterpenes with topoisomerase I. The ability of diterpenes 1 and 10 to generate DNA strand breaks in single cells was confirmed in the comet assay. However, the higher inhibitory activity on topoisomerase I of diterpenes 1 and 10 compared to CPT did not correlate with its induction of DNA damage after 1h exposition time. Diterpene 10 , but not diterpene 1 showed a similar behavior in the ability to recover from DNA strand breaks in MIA PaCa-2 cells as CPT. These differences may be due to the alkylating effects observed for diterpene 1. Although diterpene 10 exhibited a lower ability to induce DNA damage, its high dual inhibitory activity towards topo I and topo II and the observed ability to recover from the DNA strand breaks in the same range as CPT, suggested diterpene 10 to be an interesting compound for further studies. Cell cycle progression was also influenced by diterpenes 1, 4 and 10 . Diterpene 1 exhibited a significant decrease in the cell numbers at G0/G1 phase and arrest the cells in the S phase, thus inhibiting the cells to enter mitosis. Diterpenes 4 and 10 enhanced cell number in the G0/G1 phase, indicating that cells cannot enter the S phase and fail to trigger DNA synthesis. Cell cycle arrest is often mediated by alterations in cyclin-dependent kinases (CDKs) activity. However, diterpenes 1 and 10 did not exhibit significant activity against the main CDK-cyclin complexes directly involved in driving the cell cycle like CDK1/CycB1, CDK2/CycE, CDK3/CycE, CDK4/CycD3, CDK6/CycD1, CDK7/CycH/MAT1, CDK8/CycC, CDK9/CycT and PCTAIRE1. 184 Summary
Interestingly, the diterpene 2 - 4 and 10 did not induce apoptosis in MIA PaCa-2 cells, except of 1 for which slight effects were observed. Despite its quinone structure diterpene 1 failed to generate intracellular reactive oxygen species. Consequently, ROS was also not involved in the cytotoxic effects. However, diterpenes 1 - 4 and 6 - 10 exhibited high inhibitory activity towards p38 α, the mitogen-activated protein kinase which is involved in multiple pathways controlling a wide range of cellular responses, such as proliferation, differentiation and apoptosis. Further studies have to reveal whether inhibition of p38 α has any consequences on the above mentioned effects. Altogether, this work has enhanced our knowledge not only on the secondary metabolites of Peltodon longipes , but also on the biological activity of their diterpenes. Studies on their cytotoxic activity revealed new insights in structure activity relationships and in the complex molecular mechanism.
Materials and Methods 185
5. Materials and Methods
5.1. Materials
5.1.1. Chemicals
The chemicals used in the phytochemical and biological studies are described in Table 5.1.
Table 5.1 - List of used chemicals. Chemical Supplier 4-(4-nitrobenzyl) pyridine (NBP) Sigma, Steinheim Ac-DEVD-AMC (Ac-Asp-Glu-Val-Asp-7-Amino-4- Alexis, Lörrach methylcoumarin, caspase-3 substrate) Acetone Merck, Darmstadt Acetonitrile Roth, Karlsruhe Acrylamide solution, aqueous (30 % AA, 0.8 % Bis) Roth, Karlsruhe Actinomycin D Alexis, Lörrach Aprotinin Sigma, Steinheim Boric acid Roth, Karlsruhe BSA (Bovine serum albumine) Roth, Karlsruhe BSA (Bovine serum albumine, cell culture tested) Sigma, Steinheim Butylhydroxyanisole (BHA) Sigma, Steinheim Camptothecin Merck, Darmstadt Cell death detection ELISA plus - Kit Roche, Mannheim Citric acid Roth, Karlsruhe Cytochalasin B Sigma, Steinheim
Chloroform d4 (CDCl 3) Deutero GmbH, Kastellaun Collagen type I Sigma, Steinheim Dexamethasone Sigma, Steinheim Dextran T 500 Amersham, Sweden Dichlorofluorescin (DCF) Sigma, Steinheim
Dichloro-dihydrofluorescin diacetate (H 2DCFDA) Sigma, Steinheim Dichloromethane Sigma, Steinheim 186 Materials and Methods
Di-Sodium hydrogen phosphate 2-hydrate (Na 2HPO 4*2H 2O) Merck, Darmstadt Dimethylsulfoxide (DMSO) Fluka, Buchs, Sweden Dithiothreitol (DTT) Roth, Karlsruhe Dulbecco’s modified Eagle’s medium (DMEM) Gibco-BRL, Groningen Ethylenediaminetetraacetic acid (EDTA) Roth, Karlsruhe Ethylacetate Fisher Scientific, Schwerte Ethidium bromide Sigma, Steinheim Ethanol Roth, Karlsruhe Etoposide Sigma, Steinheim FBS (Fetal bovine serum) Sigma, Steinheim Ficoll-Paque TM Plus GE Healthcare Bio-sciences AB, Sweden Formic acid Roth, Karlsruhe Glucose monohydrate Merck, Darmstadt Hepes Buffer stock solution Roche, Mannheim n-Hexane Fisher Scientific, Schwerte Hydrochloric acid 37% Merck, Darmstadt Isopropyl alcohol Merck, Darmstadt Low melting point agarose Roth, Karlsruhe Leupeptin Roche, Mannheim Methanol Fluka, Buchs, Schweiz Mitomycin C Roth, Karlsruhe MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl- Roth, Karlsruhe tetrazoliumbromid) Nonidet P-40 (NP-40) Sigma, Steinheim N-Lauroylsarcosine sodium salt Sigma, Steinheim N,N- Dimethylformamid (DMF) Merck, Darmstadt Normal melting point agarose Roth, Karlsruhe p-Benzoquinone Sigma, Steinheim p-anysaldehyde Sigma, Steinheim Paraformaldehyde (PFA) Roth, Karlsruhe Penicillin-streptomycin Roche, Mannheim Pepstatin A Roche, Mannheim Petroleum ether Roth, Karlsruhe Phenylmethylsulfonylfluoride (PMSF) Sigma, Steinheim Potassium chloride Roth, Karlsruhe Materials and Methods 187
Potassium phosphate monobasic Riedle-DE HAEN, Seelze Propidium iodide Sigma, Steinheim Proteinase K Sigma, Steinheim Prolong Gold® antifade reagent with 4',6-diamino-2- Invitrogen, Germany phenylindole (DAPI) QVD-OPH, pan-caspase inhibitor MP Biomedicals RPMI 1640 (Roswell Park Memorial Institute medium) Gibco BRL, Karlsruhe SB203580 Sigma, Steinheim Sodium acetate Roth, Karlsruhe Sodium chloride Roth, Karlsruhe Sodium dodecylsulfate (SDS) Roth, Karlsruhe Sodium hydroxide Roth, Karlsruhe Sodium diphosphate-decahydrate Sigma, Steinheim Suc-Ala-Ala-Val-pNA (SAAVNA) Bachem, Switzerland Sucrose Roth, Karlsruhe TEMED (N, N, N, N-Tetramethylethylenediamine) Sigma, Steinheim Trisodium phosphate-dodecahydrate Roth, Karlsruhe Tris (Tris-(hydroxymethyl)-aminomethane) Roth, Karlsruhe Triton X-100 Sigma, Steinheim Trypan blue Sigma, Steinheim Trypsin-EDTA 0.25 % Gibco BRL, Karlsruhe Tween-20 Roth, Karlsruhe Urea Merck, Darmstadt
5.1.2. Cytokines, antibodies, enzymes and kits
The Table 5.2 contains a list of applied cytokines, antibodies, enzymes and kits and the corresponding suppliers used in this work.
Table 5.2 - Cytokines, antibodies, enzymes and kits used in this work.
Cytokine, Primer, Enzyme and Kit Company
Activating Transcription Factor 2 (ATF -2) Upstate cell signaling solutions, USA
Bradford Quick Start™ Dye Reagent 1x Bio-Rad, USA 188 Materials and Methods
Cell death detection ELISA plus Roche, Mannheim
Human DNA Topoisomerase I Inspiralis, UK
Human DNA Topoisomerase II Inspiralis, UK
Immulon Microtiter® Plates 96 wells Thermo Electron Corporation, USA
Lipopolysaccharide Sigma, Steinheim
Neutrophil Elastase (EC 3.4.21.37) Sigma, Germany
p38 α Prof. Dr. Schultz, Univ. Tübingen, Germany
Platelet Activating Factor (PAF) Bachem, Switzerland
Platelet-derived growth factor (PDGF) Invitrogen, Germany
PMA (Phorbol-12-myristate-13-acetate) Calbiochem, Germany
Phospho-ATF-2 Antibody(Thr 69/71 ) Cell Signaling Technology Inc., USA
Ribonuclease A, RNAse A Sigma, Steinheim
Supercoiled plasmid pBR322 DNA Inspiralis, UK
Tumor necrosis factor-α, recombinant human R&D Systems, Minneapolis, USA
T4-Polynucleotide kinase (10x kinase-buffer) New England Biolabs, USA
TNF-α (Tumor necrosis factor-α) RnD Systems, Germany
5.1.3. Consumable
Table 5.3 - List of used consumables. Consumable Manufacturer
0.2, 0.5, 1.7 and 2 ml reaction tubes Roth, Karlsruhe
0.2 and 1.7 ml Muti ® reaction tubes, RNase free Roth, Karlsruhe
15 and 50 ml tubes Greiner, Frickenhausen
6-, 12-, 24- and 96-well cell culture plates Greiner, Frickenhausen
96-well micro plate, Microamp ®, flat bottom Greiner, Frickenhausen Materials and Methods 189
96-well micro plate, Fluotrac 200, black, flat bottom Greiner, Frickenhausen
Aluminium foil Roth, Karlsruhe
Cell scraper 24 cm Biochrom, Berlin
Cover glasses for Neubauer chamber Menzel, Braunschweig
Coverslips glasses 10mm Zitt-Thoma, Freiburg
Cryo vials Roth, Karlsruhe
Disposal bags for autoclaving Roth, Karlsruhe
Latex gloves rotiprotect Roth, Karlsruhe
Lichrospher Si 60 (12µm), silica gel Merck, Darmstadt
Luna C-18 column (150 × 4.6 mm, 3 m) Phenomenex, Torrance
Microfuge ® tube for ultracentrifuge Beckman Coulter, Krefeld
Microscopic slides Roth, Karlsruhe
Micro-Spin™ G-25 column Amersham, Freiburg
MutiGuard barrier tips, different sizes Sorensen, Salt Lake City
Omnifix ®-F 1 ml syringe Braun, Melsungen
Parafilm Roth, Karlsruhe
Pasteur pipette Roth, Karlsruhe
Petri dishes Roth, Karlsruhe
Pipette tips, different sizes Roth, Karlsruhe
Disposable pipette with tip, 10 and 25 ml Greiner, Frickenhausen
Rotilabo ® sterile filter (0.22 µm) Roth, Karlsruhe
Seesand Roth, Karlsruhe
Silica gel 60 (0,063 – 2 mm) Merck, Darmstadt
Sterican ® 20G hypodermic needle Braun, Melsungen
Tape autoclaving sensible Roth, Karlsruhe
Tissue flasks 75 and 175 cm² Greiner, Frickenhausen
TLC Silica gel 60, aluminium sheets Merck, Darmstadt
190 Materials and Methods
5.1.4. Equipment
Table 5.4 - List of used equipment. Equipment Manufacturer
Assistent RM 5 tube agitator Karl Hecht, Sondheim
Autoclav 5075 ELV Systec, Wettenberg
Automatic Fraction Collector Retreiver II Teledyne Isco, USA Mettler-Toledo, Greifensee, Sartorius, Balances Switzerland CAMAG, automatic TLC sampler ATS 4 CAMAG, Switzerland
CAMAG, Reprostar3 Photo documentaion CAMAG, Switzerland
Centrifuge Biofuge fresco Heraeus, Hanau
Centrifuge Eppendorf 5417R Eppendorf, Hamburg
Centrifuge Rotina 35R Hettich, Tuttlingen
Clean Air work bench CleanAir Techniek, Netherlands
Electrophoresis power supply EPS 601 Amersham Biosciences
FACScaner flow cytometry system Becton Dickinson, Franklin Lakes
Fluorescence microscope Axiovert 200 Carl Zeiss, Jena
Freeze dryer Alpha 2-4 LSC Christ, Germany
Freezer VIP series -86° C Sanyo, San Diego
Gel chamber (EMSA) Thoma, Freiburg
Gel dryer 583 Biorad, Hercules
Hera cell incubator Heraeus, Hanau
Heating oven Heraeus, Hanau
HPLC - HP1090 series II (HPLC) Hewlett-Packard, Palo Alto
Ice machine Scotsman Ice Systems, Mailand,
Laboport ® mini laboratory pump KNF Neuberger, Freiburg
Liquid nitrogen tank Arpege 110 AirLiquide KGW Isotherm Karlsruhe
Low pressure liquid chromatography system (LPLC) Waters, Milford
Magnetic stirrer IKAMAG RCT IKA Labortechnik, Staufen
Mass spectrometer, TSQ 700 and LCQ-Advantage Thermo Fisher, Waltham Materials and Methods 191
Microplate Reader Modell 550 Biorad, Hercules
Microplate Reader Modell 680 Biorad, Hercules
Microplate Reader Fluostar Optima BMG, Labtech, Offenburg
Microscope Nikon TMS Nikon, Japan
Millipore water supply MilliQ Plus PF Millipore, Bedford
Multichannel pipettes Discovery, different sizes Abimed, Langenfeld
NMR Bruker DRX 400 MHz Bruker, Bremen
Neubauer cell counter chamber Paul Marienfeld, Lauda-Königshofen
Open column chromatography, different sizes Roth, Karlsruhe pH electrode Inlab ® Micro Mettler-Toledo, Greifensee pH meter Schott, Hofheim
PhosphoImager FLA-3000 Fujifilm Life Science, Stanford Eppendorf, Hamburg Pipettes, different sizes Gilson, Middletown Pipetboy acu IBS Integra Bioscience, Chur
Power Supply PowerPac 200 Biorad, Hercules
Refrigerators Liebherr, Biberach
Rotavapors Vaccubrand, Werthein
ThermoForma Steri-Cycle incubator Thermo Life Science, Freiburg
Thermomixer comfort Eppendorf, Hamburg
Thermoplate Desaga, Wiesloch
Flat bottom chambers for TLC CAMAG, Switzerland
UltraSonic bath Bandelin, Berlin
UV - detector Labomatic, Switzerland
Vortex Lab dancer Roth, Karlsruhe
Vortex Genie-2 Scientific Industries, Bohemia
Water bath GFL 1083 GFL, Burgwedel
Water bath HB4 digital IKA Labortechnik, Staufen
192 Materials and Methods
5.1.5. Plant material
Leaves, roots, aerial parts and flowers from 38 medicinal plants (Table 5.5) from different families were used in the screening phase. The plants were collected in South Brazil from March to July 2007 and authenticated by the botanist Dr. Gilberto Dolejal Zanetti. Voucher specimens are deposited in the herbarium of the Department of Biology at Federal University of Santa Maria, Brazil. Roots of Peltodon longipes A. St. Hill. ex Benth. were collected in Santa Maria, Rio Grande do Sul, South Brazil in December 2007 and were identified by the botanist Dr. Gilberto Dolejal Zanetti. A voucher specimen (SMDB 12333) has been deposited in the herbarium of the Department of Biology at Federal University of Santa Maria, Brazil.
Table 5.5 - Selected plants used in the screening phase. Species Popular name Part used
Sida rhombifolia Guanchuma Roots
Cecropia catarinensis Embaúba Leaves
Echinodorus grandiflorus Chapéu-de-couro Leaves
Cordia americana Guajuvira Leaves
Erythroxylum argentinum Cocção Leaves
Myrocarpus frondosus Cabreúva Bark
Bauhinia forfificata Pata-de-vaca Leaves
Caesalpina ferrea Pau-ferro Bark
Peltodon longipes Baicurú amarelo Roots
Luhea divaricata Acoita cavalo Leaves
Parapiptadenia rigida Angico vermelho Bark
Petiveria alliaceae Guiné Leaves
Brugmansia suaveolens Trombeteira Leaves
Schinus mole Aroeira-mansa Leaves
Gochnatia polymorpha Cambará-do-mato Leaves and Bark
Adiantopsis chlorophylla Samambaia-do-talo-roxo Leaves
Dodonae viscosa Vassoura-vermelha Leaves
Stachytarpheta cayennensis Gervão Leaves Materials and Methods 193
Vermonia tweediana Baker Assa-peixe Leaves
Mirabilis jalapa Maravilha Leaves and flower
Xanthium cavallinesii Carrapicho Leaves
Piper gaudichaudianum Pariparobão Roots
Pluchea sagitalis Erva-lucera Leaves
Alternanthera ficoidea Rabo-de-gato Aerial parts
Phrygillanthus acutifolius Erva-de-passarinho Leaves
Leonorus sibiricus Erva-de-macae Aerial parts
Leonotis nepetafolia Cordão-de-frade Flower
Irisinea herbstii Irisinea/Mussurú Aerial parts
Eupatorium laevigatum Erva-de-santana Leaves
Coleus barbatus Boldo africano Leaves
Eubrachyon ambiguum Erva-de-passarinho Aerial parts
Waltheria douradinha Douradinha Whole plant with flowers
Kalanchoe tubiflora Bálsamo-brasileiro Leaves
Jaranda micrantha Caroba Bark
Galinsoga parviflora Picão branco Aerial parts
Hedychium coronarium Falso gengibre Root hairs
Piper regnelli Pariboraba Leaves
Dichorisandra thyrssiflora Cana-de-macaco Aerial parts
5.1.6. Computer Software’s and Databases
The following software’s and database were used: HyperChem pro 6.0, Discovery Studio Visualizer 2.5, Origin, WinCats (CAMAG), ChemDraw Ultra 8.0, Corel Photo Paint, Cell C software, MestreNova, Schrödinger Suite 2009 Induced Fit Docking protocol; Glide version 5.5, Scifinder Scholar, Beilstein Crossfire, Pub med and ISI web of knowledge.
194 Materials and Methods
5.2. Methods
5.2.1. Chromatographic and spectroscopic methods
Thin Liquid Chromatography (TLC) TLC analyses were used for monitoring the fractionation and purification of the diterpenes. Aluminum plates (20 x 20 cm) coated with Silica gel 60 F 254 as adsorbent were used. In general 10 to 25 µl of each probe were applied bandwise onto the plates using the automated TLC sampler (Camag, Switzerland). TLC plates were placed in a developing chamber and eluted with different mixture of solvents and dried at 110°C on a thermoplate. Plates were derivatized with anisaldehyde-sulphuric reagent and photographed at white lamp, and UV at 254 nm and 366 nm. TLC eluents (v:v) used: 1 - EtOAc: n-hexane (35:75) - Fractionation of filtrate part.
2 - petroleum ether:CH 2Cl 2:EtOAc (9.0:0.5:0.5) – Subfractionation of F1 and RF2.
3 - petroleum ether:CH 2Cl 2:EtOAc (5.0:4.6:0.4) – Subfractionation of F8.
4 - petroleum ether:CH 2Cl 2:EtOAc (4.5:5.0:0.5) – Subfractionation of F11.
5 - petroleum ether:CH 2Cl 2:EtOAc (3:6:1) – Subfractionation of F13 and F14. 6 - petroleum ether:EtOAc (20:3) – Fractionation of residue
7 - petroleum ether:CH 2Cl 2:EtOAc (8:1:1) - Subfractionation of RF2.SF4 and RF2.SF5 The anisaldehyde-sulphuric reagent was prepared by adding carefully 10 ml of sulphuric acid to an ice-cold mixture of 170 ml methanol and 20 ml acetic acid. Finally, 1 ml of anisaldehyde was added. The reagent was sprayed on TLC plates, which were then heated at 110°C until color development.
Open Column Chromatography (OC) Open column chromatography with 50 cm long and 3 cm in diameter was used in the fractionation of Peltodon longipes. The OC was homogenously packed with silica gel 60 (0.0630 – 2.000 mm) (Merck) as stationary phase.
Materials and Methods 195
Low Pressure liquid Chromatography (LPLC) LPLC system was employed for fractionation of the main fractions obtained from the n- hexane extract of Peltodon longipes by OC. Elution of mobile phase was performed using a binary pump, model S 1990 (Waters-Millipore) connected to a closed glass column (different sizes) filled with silica gel Lichrospher® Si 60 (12 µm) (Merck). Elution and separation was monitored by an UV detector (Labomatic) at 275 nm connected to a chart recorder BBC Goerz Metrawatt SE 120 .
High Pressure liquid Chromatography (HPLC) HPLC analysis was carried on a HP-1090 system (Hewlett–Packard, Palo Alto, CA), equipped with autosampler, tertiary pump and photodiode array-detector using a Phenomenex Luna reversed phase column (C18; 3 µm; 150×4.6 mm). A security guard cartridge (4.0 x 3.0 mm I.D.) was used to protect the analytical column. The following eluents were used; mobile phases A (H 2O-ACN, 95:5) and B (ACN-H2O, 95:5), both with 0.1% HCOOH; linear gradient starting with 60% of B, increasing to 100% at 10 min until 20 min and re-equilibration of the column from 20 to 25 min with 60% of B; flow rate 0.8 ml/min, detection at λ 275 nm; sample injection of 10 µl.
The quantification of the main isolated compound (7α-acetoxyroyleanone) in the crude extract of Peltodon longipes was performed using the calibration curve. Stock solutions of 200 µg/ml 7 α-acetoxyroyleanone in acetonitrile (HPLC grade) were prepared in 10 ml volumetric flasks. Calibration samples were prepared by dilution of the stock solution with acetonitrile, to obtain five solutions ranging from 1 to 200 µg/ml. Each measurement was carried out in three replicates to verify the reproducibility of the detector response at each concentration level. The peak areas of the chromatograms were plotted against the concentrations of 7 α-acetoxyroyleanone to obtain the calibration curve. The five solutions were subjected to regression analysis to calculate calibration equation and correlation coefficients. A concentration of 500 µg/ml of the n-hexane extract was used to calculate the amount of 7 α-acetoxyroyleanone in the extract of Peltodon longipes .
196 Materials and Methods
Mass Spectroscopy (MS) MS data were taken with the following instruments: Electron Impact (EI) and Chemical Ionization (CI-MS) mass spectra were recorded by the direct injection of the sample in a TSQ 700 mass spectrometer (Thermo Fisher, Waltham) with a Triple-Quadrupole analyzer at 70 eV ionization energy. Electrospray (ESI) and Atmospheric Pressure Chemical Ionization (APCI) mass spectra were recorded in a LCQ-Advantage mass spectrometer (Thermo Fisher, Waltham) with an Iontrap. High Resolution Electron Impact Ionization mass spectra (HREI- MS) analyses were done in a MAT-95XL double-focusing magnetic field mass spectrometer (Thermo Fisher).
Nuclear Magnetic Resonance Spectroscopy (NMR) The isolated and purified compounds were elucidated based on 1D-(1H and 13 C) and 2D (COSY, HSQC and HMBC) nuclear magnetic resonance spectroscopy experiments. NMR spectra were recorded in CDCl 3 on a Bruker DRX 400 MHz instrument (Bruker, Bremen, Germany) at 400 MHz ( 1H) and 100 MHz ( 13 C). The chemical shifts ( δ) are in ppm and the coupling constants ( J) in Hertz (Hz).
Liquid Chromatography with Nuclear Magnetic Resonance Spectroscopy (LC-NMR) The preliminary identification of target components at the earliest stage of separation is a strategic element in guiding selective isolation procedures. The introduction of on-line solid- phase extraction (SPE) coupled to high performance liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) is considered a powerful method for studying complex mixtures such as crude plant extracts. Therefore, in cooperation with Prof Dr .J.W. Jaroszewski (Dept. Medicinal Chemistry, University of Copenhagen, Denmark) studies on the crude n-hexane extract of Peltodon longipes was carried out (Lambert et al. 2005; Clarkson et al. 2005). The HPLC-SPE-NMR instrument consisted of a Bruker LC22 quaternary solvent delivery pump with Degasys Populaire degasser, and an Agilent 1100 autosampler, a Bruker DAD UV detector, a Knauer K100 Wellchrom pump for postcolumn water delivery, a Spark Prospekt 2 solid-phase extraction device, and a Bruker Avance 600 spectrometer (hydrogen frequency 600.13 MHz) equipped with a 30 µl inverse 1H ( 13 C) flow-probe operating at 25°C. All analytical-scale HPLC separations was performed on a 150 x 4.6 mm i.d., 3 µm particle size, Phenomenex Luna C 18 (2) column with a precolumn. Chromatography, peak trapping, Materials and Methods 197 and analyte transfer from the SPE unit to the NMR spectrometer were controlled with HyStar ver. 2.3 software, and NMR experiments were conducted with Xwin-nmr ver.3.1 software (Bruker BioSpin).
Ultraviolet-visible Spectroscopy (UV-Vis) Ultraviolet absorption spectra of each isolated compound were obtained using the photodiode array detector in the HP-1090 system (Hewlett-Packard, Polo Alto, CA).
5.2.2. Plant Extraction and Isolation Methods
Brazilian medicinal plants used in the screening phase The selected parts of each plant (Table 5.5) from different families used in the screening phase were dried, except of Sedum dendroideum and Kalanchöe tubiflora , grounded and extracted using soxhlet, ultrasound or maceration. The soxhlet extraction was performed with 25 g of plant material, using at first n-hexane (250 ml), and after drying, ethanol (250 ml). 10 g were taken for the ultrasound extraction using n-hexane (100 ml) followed by ethanol (100 ml). The maceration process was carried out with 438.5 g of Sedum dendroideum and 329.5 g of Kalanchöe tubiflora . Each solvent was applied twice directly to the ground plant material during 16 days changing the solvent each 8 days. Firstly, n-hexane was used for 16 days followed by ethanol with the same material for another 16 days. The solvents were removed under vacuum at 40°C. Finally, extracts were lyophilized.
European medicinal plants Flowers from Calendula officinalis and Matricaria recutita were provided by Heinrich GmbH & Co., Schwebheim, Germany. Flowers of the respective plants were powdered and 10 g extracted in a soxhlet apparatus using first n-hexane and subsequently ethanol (150 ml each). The solvent was removed by a vacuum evaporator at 40°C and lyophilized. The following amounts were obtained: Calendula : 1.29 g n-hexane and 1.89 g ethanolic extract; Matricaria recutita : 0.65 g n-hexane and 1.12 g ethanolic extract. The extracts were stored in sealed vials at -20ºC before used for further analysis. Faradiol myristate acid ester and 198 Materials and Methods faradiol palmitate acid ester were isolated from Calendula flowers and kindly provided by Prof Dr. Hamburger (Hamburger et al. 2003).
Peltodon longipes The air-dried and ground roots (553 g) of Peltodon longipes were exhaustively extracted using a soxhlet apparatus with n-hexane (500 ml). After extraction the solvent was removed under vacuum and lyophilized, affording a crude n-hexane extract (27 g). The deffating process was carried out by dissolving the extract in 500 ml methanol and placed it in the refrigerator at - 20°C for 48h. Subsequently, the solution was filtered affording two parts which were kept separately and named as residue (5.2 g) and filtrated part (18.5 g). 3 g of the filtrated part was dissolved in ethylacetate, adsorbed onto silica gel (3 g), dried and subjected to open column chromatography (50 x 3 cm) with silica gel 60 (0.063-0.200 mm) (150 g). Elution started with n-hexane and increasing polarity with ethylacetate at a flow rate of 1 ml/min. The fractions were collected in reaction tubes with approximately 10 ml each resulting in 1013 tubes. The fractions were screened by TLC (Figure 5.2) and HPLC (Figure 2.7) and those with similar composition were combined to give a total of 15 fractions (F1-F15) (Figure 5.1 and Table 5.6).
Materials and Methods 199
553 g roots of P. longipes
Soxhlet extraction n-hexane
27 g of lipophilic extract
Fat removing MeOH -20°C
5.2 g 18.5 g Residue Filtrate
Fractionation of 3 g Fractionation of 3 g by OC with silica gel by OC with silica gel 60 60 4 Fractions 15 Fractions RF1 – RF4 F1 – F15
LPLC
RF2 RF3 F1 F8 F11 F13 F14 Further chromatographed by LPLC using silica gel column LPLC ( 1) (LiChrospher® Si 60 (12µm)) ( 11) (12)
(3) ( 5) (7) (9) ( 2) (8) (6) ( 10 ) ( 4)
Figure 5.1 - Extraction and isolation scheme of the diterpenes from the n-hexane extract of Peltodon longipes .
Table 5.6 – Amounts of each fraction obtained by fractionating the n-hexane extract (filtrate part) of Peltodon longipes . Fractions Amount (mg)
F1 265.8
F2 35.1
F3 21.4
F4 35.1
F5 17.5
F6 44.5
F7 25.9
F8 414.6
F9 307.7
F10 63.9 200 Materials and Methods
F11 172.5
F12 70.5
F13 172.1
F14 381.8
F15 401.2
Figure 5.2 - TLC of the main fractions (F1 to F15) obtained during fractionation by OC of the filtrated part from the n-hexane extract of P. longipes . TLC silica gel plates were eluted with EtOAc: n-hexane (35:75) and detection was performed with anisaldehyde-sulphuric reagent.
Further separations of the selected fractions from the n-hexane extract (filtrated part) of P. longipes were carried out on a LPLC system using a silica gel column (LiChrospher® Si 60 (12µm)) with different sizes and different mobile phases as described below. To monitor the separation and the purity of the isolated compounds TLC and HPLC were employed. Fraction 1 (F1) (265.8 mg) was chromatographed at a flow rate of 0.7 ml/min using petroleum ether:CH 2Cl 2:EtOAc (9.0:0.5:0.5) mixture as eluent. The same eluent was used for TLC monitoring. Six subfractions (F1.SF1 - F1.SF6) were obtained, (see Table 5.7). F1.SF1 yielded compound 3 (42.8 mg), F1.SF4 5 (12.1 mg), F1.SF5 7 (6.2 mg) and F1.SF6 9 (11.2 mg). Materials and Methods 201
Table 5.7 - Amounts of each subfraction obtained after chromatographic separation of F1. F1 - SubFractions Amount (mg)
F1-SF1 42.8 ( 3)
F1-SF2 88.2
F1-SF3 25.1
F1-SF4 12.1 ( 5)
F1-SF5 6.2 ( 7)
F1-SF6 11.2 ( 9)
Fraction 8 (F8) (414.6 mg) was chromatographed with petroleum ether:CH 2Cl 2:EtOAc (5.0:4.6:0.4) at a flow rate of 0.45 ml/min affording 5 subfractions (F8.SF1 – F8.SF5) (see Table 5.8). The same solvent was used for TLC monitoring. F8.SF5 yielded 2 (65.5 mg).
Table 5.8 - Amounts of each subfraction obtained after chromatographic separation of F8. F8 - SubFractions Amount (mg)
F8-SF1 18.7
F8-SF2 93.4
F8-SF3 53.7
F8-SF4 59.2
F8-SF5 65.5 ( 2)
Fraction 11 (F11) (172.5 mg) was chromatographed at a flow rate of 0.45 ml/min using a mixture of petroleum ether:CH 2Cl 2:EtOAc (4.5:5.0:0.5) as eluent. The separation afforded 4 subfractions (F11.SF1 – F11.SF4) (see Table 5.9). F11.SF3 yielded 8 (10.1 mg) and F11.SF4 yielded 6 (17.5 mg). The same solvent was used for TLC monitoring.
Table 5.9 - Amounts of each subfraction obtained after chromatographic separation of F11. F11 - SubFractions Amount (mg)
F11-SF1 55.0
F11-SF2 22.3
F11-SF3 10.1 (8)
F11-SF4 17.5 (6)
202 Materials and Methods
Fraction 13 (F13) (172.1 mg) was chromatographed with petroleum ether:CH 2Cl 2:EtOAc (3:6:1) at a flow rate of 0.7 ml/min. The separation afforded 5 subfractions (F13.SF1 – F13.SF5) (see Table 5.10). F13.SF3 afforded 10 (11.9 mg). The same solvent was used for TLC monitoring.
Table 5.10 - Amounts of each subfraction obtained after chromatographic separation of F13. F13 - SubFractions Amount (mg)
F13-SF1 13.1
F13-SF2 5.0
F13-SF3 11.9 ( 10 )
F13-SF4 4.2
F13-SF5 12.1
Fraction 14 (F14) (381.8 mg) was chromatographed at a flow rate of 0.9 ml/min using petroleum ether:CH 2Cl 2:EtOAc (3:6:1) as eluent. The separation afforded 6 subfractions (F14.SF1 – F14.SF6) (see Table 5.11). F14.SF1 gave 4 (10.5 mg). The same solvent was used for TLC monitoring.
Table 5.11 - Amounts of each subfraction obtained after chromatographic separation of F14. F14 - SubFractions Amount (mg)
F14-SF1 10.5 (4)
F14-SF2 12.1
F14-SF3 61.4
F14-SF4 20.2
F14-SF5 4.6
F14-SF6 29.5
The residue part (3 g) was first chromatographed by open column chromatography (50 x 3 cm) with silica gel 60 (0.063-0.200 mm) (150 g) using CH 2Cl 2:MeOH:EtOAc (9.50:0.25:0.25) as eluent at a flow rate of 2 ml/min. The fractions were collected in reaction tubes with approximately 15 ml each resulting in 350 tubes. The fractions were screened by TLC using petroleum ether:EtOAc (20:3) as eluent and those with similar composition were Materials and Methods 203 combined to give a total of 4 fractions (RF1 – RF4). RF3 yielded compound 1 (686.8 mg). (See Table 5.12 and Figure 5.3).
Table 5.12 - Amount of each fraction obtained through fractionation of the n-hexane extract (residue part) form the roots of Peltodon longipes. Fractions Residue Amount (mg)
RF1 224.9
RF2 354.2
RF3 686.8 ( 1)
RF4 316.7
Residue RF1 RF2 RF3 RF4
Figure 5.3 - TLC of the main fractions (RF1 to RF4) obtained during fractionation by OC of the residue part from the n-hexane extract of P. longipes . TLC silica gel plate was eluted with petroleum ether:EtOAc (20:3) and detection was done with anisaldehyde-sulphuric reagent.
Fraction 2 from residue (RF2) was further chromatographed on silica gel column
(LiChrospher® Si 60 (12µm)) by LPLC with petroleum ether:CH 2Cl 2:EtOAc (9.0:0.5:0.5) at a flow rate of 0.9 ml/min. The separation afforded six subfractions (RF2.SF1 – RF2.SF6) (see Table 5.13). RF2.SF1 yielded 12 (5.3 mg). The same solvent was used for TLC monitoring. 204 Materials and Methods
Table 5.13 - Amounts of each subfraction obtained after chromatographic separation of RF2. RF2 - SubFractions Amount (mg)
RF2.SF1 5.3 (12 )
RF2.SF2 46.5
RF2.SF3 31.4
RF2.SF4 58.1
RF2.SF5 12.5
RF2.SF6 37.1
RF2.SF4 and RF2.SF5 (70.6 mg) were combined and rechromatographed using the same system as RF2 with petroleum ether:CH 2Cl 2:EtOAc (8:1:1) at a flow rate of 0.45 ml/min. The separation afforded 3 subfractions (R2-SSF1 – R2-SSF3) (see Table 5.14). RF2.SSF1 afforded 11 (4 mg). The same solvent was used for TLC monitoring.
Table 5.14 - Amounts of each subfraction obtained after chromatographic separation of RF2.SF4 combined with RF2.SF5. RF2.SF4 and RF2.SF5 - SubFractions Amount (mg)
RF2.SSF1 4.0 (11 )
RF2.SSF2 14.5
RF2.SSF3 23.1
5.2.3. Biological Methods
5.2.3.1 Cell culture
Cultivation of adherent cells Swiss 3T3 albino mouse fibroblasts, human pancreatic cancer cell line (MIA PaCa-2) and human melanoma cancer cell line (MV-3) were used in this work (Table 5.15). Materials and Methods 205
Table 5.15 - List of used cells and their respective culture medium composition and supplier.
Cells Medium Supplements Supplier 10% FCS, 100 µg/ml Swiss 3T3 albino DMEM (4.0 g/l Cell Line Service, penicillin, 100 µg/ml mouse fibroblasts glucose) Appelheim, Germany streptomycin 10% FCS, 100 IU/ml RPMI-1640 (2.0 g/l ATCC number MIA PaCa-2 penicillin, 100 µg/ml glucose) CRL-1420 TM streptomycin 10% FCS, 100 µg/ml Dr GP van Muijen (Dept. of DMEM (4.0 g/l MV-3 penicillin, 100 µg/ml Pathology, Nijmegen, glucose) streptomycin Netherlands)
Swiss 3T3 albino mouse fibroblasts Swiss 3T3 albino mouse fibroblasts (Cell Line Service, Appelheim, Germany) was kindly supplied by Dr. J. Orth (Institute of Experimental Pharmacology and Toxicology, University of Freiburg, Germany) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin, at 37ºC in a humidified atmosphere containing 5% CO 2 (all Gibco-BRL, Netherlands). Fibroblasts were used in monolayers for evaluation of the wound reepithelialization potential of crude herbal extracts and isolated compounds.
Human pancreatic cancer cell line (MIA PaCa-2) MIA PaCa-2 cell was obtained from the American Type Culture Collection (ATCC number CRL-1420 TM , Manassas, USA) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin, at 37ºC in a humidified atmosphere containing 5% CO 2 (all Gibco-BRL, Netherlands).
Human melanoma cancer cell line (MV-3) The MV-3 cell line was kindly supplied by Dr GP van Muijen (Dept. of Pathology, Nijmegen, Netherlands) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin, at 37ºC in a humidified atmosphere containing 5% CO 2 (all Gibco-BRL, Netherlands). 206 Materials and Methods
Every 2 - 3 days when the cells have reached a confluent monolayer, they were harvested from the tissue-culture flasks with trypsin-EDTA 0.25%, suspended in appropriate number for cultivation and/or plated for each experiment at a desired cell density.
Cell counting, stimulation and treatment For counting the cells, a cell suspension was gently stirred and 5 µl added to 15 µl of trypan blue solution and mixed. 10 µl was then pipetted to a Neubauer chamber and cells were counted in all 4 quadrants. The sum of the 4 quadrants was then divided by four and multiplied by the dilution factor. The resulting number represents cell number x 10 4 per ml of medium. Cell concentration could then be adjusted to the required number by adding fresh medium.
Freezing and thawing of cells Cryopreservation of cell lines in liquid nitrogen at -170°C is a common method for the long-term storage of a cell reservoir. Preferably, cells were prepared for freezing at an early time point in their cultivation indicated by a low number of passages. Therefore, cells were first counted and the respective amount (usually 2 x 10 6 cells per ml freezing medium) centrifuged and washed in ice-cold PBS. The pellet was resuspended in freezing medium consisting of FCS and DMSO (9:1 v/v). Approximately 1-2 ml of the cell suspension was transferred to a cryo vial. Cryo vials were first transferred to a -80°C freezer in a lockable styrofoam box and after 2-3 days put in a liquid nitrogen tank for long-term storage. To thaw cells, the cryo vials was placed in a 37°C water bath and subsequently transferred into a culture flask with pre-warmed cultivation medium. Medium was always changed in the following day since DMSO leftovers and dead cells should be removed.
5.2.3.2 Human Neutrophil Elastase assay
For studying the effects on human neutrophil elastase (HNE), the isolation of human neutrophils and the assays were performed as previously reported by Schorr et al. 2005. The neutrophils were isolated in the morning to avoid basal neutrophil stimulation from fresh blood of healthy adult volunteers. Fresh blood (25 ml each) from two healthy volunteers (in total 50 ml) was collected in simple syringes with Na + -heparin (100 µl). The fresh blood of each donor was treated with 3 ml Dextran T500 for erythrocyte sedimentation for 40 min. The Materials and Methods 207 supernatant (15 ml) was transferred to another tube and diluted with PBS buffer up to a volume of 40 ml. Ten milliliters of Ficoll-Paque TM Plus was added and centrifuged at 500 g for 30 min at 4°C. The remaining erytrocytes were lysed with 5.5 ml of cold water for 20 s. After the addition of 44 ml of sucrose-HEPES buffer (hypertonic), the cells were centrifuged at 500 g for 10 min at 4°C. The cell pellet was suspended at a concentration of 15 x 10 6 cells/ml in PBS containing Ca 2+ /Mg 2+ . The isolated cells were mixed resulting in a cell suspension of 5.5 ml and immediately used for human neutrophil elastase release assay and the direct influence on human neutrophil elastase. Stock solutions from the extracts were prepared in DMSO, which was restricted to 0.2%. The isolated were treated either by incubation with cytochalasin B and platelet-activating factor (PAF) to release elastase and subsequent incubation of the supernatant with the test compound and enzyme substrate (direct HNE inhibition) or by incubation of the neutrophils with a solution of cytochalasin B, stimulant, elastase-substrate and the test compound (inhibition of HNE release). In both cases the reaction was stopped with citric acid and the released product was measured photometrically at 405 nm.
5.2.3.3 Scratch assay
The proliferation and migration abilities of fibroblasts exposed to medicinal plant extracts and single compounds were assessed using a scratch wound assay which measures the expansion of a cell population on surfaces. The assay was performed as reported in Fronza et al. 2009. Swiss 3T3 albino mouse fibroblasts were plated into 24-well tissue culture dishes containing coverslips precoated with collagen type I (40 µg/ml) for 2h at 37ºC at a concentration of 3×10 5 cells/ml and cultured to nearly confluent cell monolayers. An artificial linear wound was generated with a sterile 100 µl plastic pipette tip; cellular debris was removed by washing the coverslips with PBS. DMEM medium with DMSO (0.25%), platelet derived growth factor (2 ng/ml) and the test substances was added to set of 3 coverslips per dose and incubated for 12h at 37ºC with 5% CO 2. The cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 4',6-diamino-2-phenylindole (DAPI) overnight. Three representative images from each coverslip of the scratched areas under each condition were photographed to estimate the relative migration cells. Data were analyzed 208 Materials and Methods using CellC software (Selinummi et al. 2005). The sample values were referred to untreated control and experiments were performed at least in duplicate.
5.2.3.4 Cytotoxic studies
Viability assay using MTT Viability was assessed by the standard colorimetric assay using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide). The assay is based on the reduction of the yellow, water-soluble MTT to purple water-insoluble formazan crystals in the mitochondria of living cells. During this process, the MTT reagent is reduced by means of NADH in a succinate-dependent dehydrogenase reaction. Since this reaction can only occur in viable cells, the production of formazan crystals can directly be correlated to cell viability. Accordingly, MTT assay can give a first idea of the impact of a treatment on cell viability and provides therefore general information on cytotoxicity (Mosmann 1983).
Figure 5.4 - MTT reaction. Reduction of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide to a purple formazan. This reaction is catalyzed by a mitochondrial succinate-dependent dehydrogenase and occurs only in living cells.
The MTT assay was carried out based on the method described by Mosmann 1983. In brief, human tumor cells (MIA PaCa-2 and or MV-3) were plated in 96-well flat-bottomed tissue culture plates with 11,000 - 12,000 cells per well in 150 µl culture media followed overnight incubation at 37ºC (5% CO 2 and 95% air) to allow cell attachment. Then, cells were incubated for 24h in the presence or absence of 50 µl of increasing concentrations of the plant extract or pure compounds dissolved in DMSO. Camptothecin (CPT) and etoposide (ETO) were used as positive controls. Control cells were treated with the highest concentration of Materials and Methods 209
DMSO (0.1%) as vehicle control. Then, 100 µl of MTT solution (2.5 mg/ml in PBS:medium (1:2)) was added per well and the plate incubated for additional 3h to allow metabolism of MTT by cellular mitochondrial dehydrogenase. The excess MTT was aspirated and the formazan crystals formed were dissolved by the addition of 200 µl of extraction solution buffer (20% SDS, 50% DMF). After overnight incubation, absorbance of purple formazan, proportional to the number of viable cells, was measured at 595 nm using a microplate reader (Bio-Rad, Japan). The sample values were referred to the untreated control. The results were analyzed using GraphPad (GraphPad Prism 5) computer program. The IC 50 values with 95% confidence interval were obtained by non-linear regression (concentration versus percentage of inhibition).
MTT assay with pan-caspase inhibitor (Q-VD-OPH) The pan-caspase inhibitor (Q-VD-OPH) was used to verify the ability of tested compound to inhibit the caspase activation and therefore protect cells against drug toxicity depended of apoptosis. MTT assay with pan-caspase inhibitor (Q-VD-OPH) was performed using the same conditions as previously described in section 5.2.3.4. Pre-incubation of the cells with 25 µM Q-VD-OPH for 1 hour were included in the procedure before adding the tested compounds.
MTT assay with ROS-scavenger Butylated hydroxyanisole (BHA) is considered as a scavenger of reactive oxygen species (ROS). ROS are multifaceted signaling molecules implicated in a variety of cellular programs during physiological as well as pathological conditions. It is well known that the excessive production of ROS is hazardous for living organisms and damages major cellular constituents such as DNA, lipid and protein. Therefore, MTT assay with BHA was performed using the same conditions as previously described in section 5.2.3.4. However a pre-treatment of cell with 100 µM BHA before adding the tested compounds was included.
Cytotoxicity with propidium iodide (PI) Propidium iodide (PI) cannot enter living cells and is commonly used in protocols for identifying dead cells in a population. PI dye binds to DNA and become fluorescent. MIA PaCa-2 cells were treated for 24h in the presence or absence of increasing concentrations of test compounds. Them cells were harvested with trypsin – EDTA 0.25% and washed in cold phosphate-buffered saline, centrifugated (1500 rpm, 4°C, 5 min) and the cell pellet resuspended in 1 ml of cold PBS and finally stained with 2 µl PI (stock solution 1 mg/ml). 210 Materials and Methods
After 5 min of incubation at room temperature, the fluorescence was measured by a FACScan Cytometer (Beckton Dickinson) in the FL2 channel (575 nm).
NCI-60 DTP Human Tumor Cell Line screen
7 α-acetoxyroyleanone and royleanone were screened in the Molecular Cancer Therapeutics Program NCI-60 DTP human tumor cell line panel. This screen employ 60 different human tumor cell lines, representing leukemia, melanoma and non-small cell lung cancer, colon, brain, ovary, breast, prostate, and renal, and the assays was performed following standard methodology by Developmental Therapeutics Program (DTP) (Shoemaker 2006).
5.2.3.5 Caspase-3 like assay
Caspases are proteases that play an important role in the execution of apoptosis by controlled degradation of cell proteins. Caspase-3 and -7 belong to the executioner caspases. Their activity can be used as a quantitative measure of programmed cell death. For measuring caspase-3/-7 activity in response to different treatments with herbal medicinal plants and the respective isolated diterpenes caspase-3-like assay was performed. This assay relies on the reaction between the enzyme and the caspase-3/-7 specific substrate in a 96-well formate. The substrate is comprised of the amino acid recognition- and cutting sequence of caspase-3/-7 (Asp-Glu-Val-Asp) and is additionally linked to the fluorophore AMC (7-amino-4-methyl coumarin). After cleavage, AMC derived fluorescence can be detected and quantified as a measure for caspase-3/-7 activity (Schmich 2011). For performing caspase-3 assay, 1.5 x 10 6 cells were washed with 1 ml icecold PBS, scraped in 500 µl PBS and collected in Eppendorf tubes. After centrifugation (2150 g, 4°C, 3 min), the pellet was resuspended in 50 µl homogenization buffer (31 mM Hepes-KOH pH 7.4, 2.5 mM EGTA, 2.5 mM MgCl 2) supplemented freshly with the protease inhibitors (12 mM DTT, 120 µM PMSF, 12 ng/ml leupeptin, 500 ng/ml pepstatin, 12 µg/ml aprotinin, 1.5 µg/ml cytochalasin B). Lysis of the cells was achieved by freezing in liquid nitrogen, then thawing in a 40°C water bath and subsequent vortexing which was repeated two more times. After final centrifugation at 20800 g at 4°C for 10 minutes, 8 µl of the supernatant was added to one well of a Fluotrack 96-well plate containing 90 µl of assay buffer (100 mM Hepes-KOH pH 7.5 containing 100 mM Materials and Methods 211
DTT). 1 µl of the fluorogenic caspase-3/-7 substrate Ac-DEVD-AMC was immediately added (final concentration of 200 nM) and AMC production was monitored over 40 measurement cycles in a Fluostar Optima plate reader with an excitation wavelength of 370 nm and an emission wavelength of 450 nm. To determine relative fluorescence unit (RFU) values, increase in fluorescence over the time of 40 cycles (= slope m) was divided by the protein concentration (= c) determined by Bradford assay. Sample values were determined as fold increase referred to untreated control.
Relative fluorescence unit (RFU) sample x = m x / c x
5.2.3.6 Cell death detection ELISA
DNA fragmentation and condensation are typical hallmarks of apoptosis. Accordingly, DNA-histone complexes form mono- and oligonucleosomes which can be detected by specific antibodies. In this work, DNA fragmentation was measured by the cell death detection ELISA plus Kit (Roche Diagnostics) according to the manufacturer’s instructions. Briefly, MIA PaCa-2 cells (3.3 × 10 4 cells/ml) were incubated with test substances for 16h. After incubation time, cell suspension was centrifuged (2150 g, 4°C, 3 min) and cell lysis was performed by adding 200 µl of lysis buffer and incubation at room temperature for 30 minutes. Histone-DNA complexes in the supernatant were detected by ELISA. Therefore, 20 µl of the supernatant were transferred to a streptavidin-coated microplate together with 80 µl incubation buffer. After 2h of incubation at room temperature with 300 rpm shaking, the solution was removed; the wells were washed three times with 250 µl incubation buffer, and incubated with 100 µl of ABTS solution for up to 15 minutes at 250 rpm at room temperature in a thermomixer. 100 µl of ABTS stop solution were then pipetted to each well and absorbance measured at a wavelength of 405 nm with a reference wavelength of 520 nm. Measured values were referred to untreated cells and given as enrichment factor.
212 Materials and Methods
5.2.3.7 Quantification of ROS by dichlorofluorescin fluorescence assay
Reactive oxygen species (ROS) accumulation is a key feature of many cellular dysfunctions and is often involved in apoptosis. ROS is a general description of different reactive ions which are difficult to distinguish in the cell by biochemical methods. However, it is possible to measure the total amount of ROS by determining the oxidation capacity. In this work, ROS were assessed by the oxidation-sensitive probe 2,7-dichloro-dihydrofluorescin diacetate (H 2DCFDA). H 2DCFDA diffuses easily across the cell membrane and is then trapped in the cell by deacetylation. In the presence of ROS, H 2DCF is rapidly oxidized to the highly fluorescent compound dichlorofluorescin (DCF), which can be quantified by fluorescence measurement. This work was performed as previously described by Schmich 2011. Briefly, 7.5 x 10 5 of
MIA PaCa-2 cells were incubated with different stimuli for 24h and H 2DCFDA was added to a final concentration of 20 µM in the last 20 minutes of incubation. After scraping, centrifugation at 2150 x g, 4°C for 3 min, washing with PBS and another centrifugation (2150 g, 4°C, 3 min), cells were lysed in 70 µl lysis buffer (20 mM Tris-HCl pH 7.4, 136 mM NaCl, 2 mM EDTA, 10 % glycerol, 4 mM benzamidine, 50 mM β-glycerophosphate, 20 mM
Na 3PO 4, 1 mM Na 3VO 4) by shaking at 1400 rpm and 4°C in a Thermomixer for 20 min followed by a final centrifugation at 20800 g, 4°C for 10 min. 10 µl of cell lysate was diluted with 90 µl of assay buffer (100 mM Hepes-KOH pH 7.5 containing 100 mM DTT) in a Fluotrack 96-well plate and fluorescence was determined using an excitation wavelength of 485 nm and emission wavelength of 540 nm. To quantify fluorescence, a DCF standards ranging from 0.01 µM up to 0.75 µM were measured and a standard calibration curve calculated. Sample values were determined as fold increase referred to untreated control.
5.2.3.8 Alkylating activity
The term alkylating agent in general denotes those compounds chemically reactive capable of replacing a hydrogen atom in another molecule by an alkyl radical, and this involves electrophilic attack by the alkylating agent so that the definition must be extended to include those reaction involving addition of the radical to a molecule containing an atom in a lower Materials and Methods 213 valency state (Warwick 1963). 4-(4-nitrobenzyl)pyridine (NBP) is an analytical reagent that has been used for the estimation of concentration levels of specific alkylating agents. Accordingly to these procedure equal volumes of various known concentration levels of a given alkylating agent are heated with a large excess of the NBP reagent for a period of time; after cooling and addition of alkali, the intensity of violet color development is determined spectrophotometrically (Bardos et al. 1965; Manso et al. 2005). Alkylation activity was determined by small modifications of the procedure described by Thomas et al. 1992 and Kim & Thomas 1992. In a reaction tube 1 ml of acetone, the test compounds, 1.5 ml 0.2 M sodium acetate-acetic buffer (pH 4), 0.5 ml of 5% NBP solution in acetone (v/v), and sufficient water were added to produce a total volume of 3.5 ml. The reaction tube was placed in a 37°C water batch. After 5 min the solution was chilled on ice (2 min) and 0.5 ml of ethylacetate/acetone (5:2, v/v) added, followed by 0.5 ml 5 N sodium hydroxide and 0.5 ml ethylacetate. After mixing in a vortex for 10 s, the organic phase was separated and the absorbance measured at 540 nm. Following the addition of NaOH the absorbance was measured not longer than 2 min. The analysis was performed at least in triplicate.
5.2.3.9 Topoisomerases relaxation assays
The assays on human DNA topoisomerases were carried out by Inspiralis Limited® (Norwich, United Kingdon) according to the internal standard operating procedure (SOP). In all experiments, the activity of the human topoisomerase I and/or II was determined prior to the testing of the compounds and 1 U defined as the amount of enzyme required to fully relax the substrate (see explanation of gels Figure 5.5). Compounds were titrated in a range of concentrations from 1 nM to 1 mM. Compounds were serially diluted in DMSO with the final dilution to 10% (v/v) DMSO and added to the reaction before the addition of the enzyme. The final DMSO concentration in the assay was 10% (v/v).
214 Materials and Methods
Topo I/II - +
OC (nicked) relaxed
supercoiled
Figure 5.5 - Explanation of the gels. 1 UI of topo I and/or II enzyme were needed to relax the supercoiled plasmid DNA (pBR322).
The substrate used (pBR322) consisted of a dominant supercoiled band with a faint trace of nicked open circular DNA above. The relaxed DNA consisted of a range of topoisomers which ran as a series of bands close to the nicked band. Treatment of supercoiled pBR322 with topo I and/or II converts the single supercoiled band to the relaxed range of topoisomers as seen in Figure 5.5.
Topoisomerase I Relaxation assay 1 U of topo I was incubated with 0.5 g supercoiled plasmid DNA (pBR322) in a 30 l reaction at 37 °C for 30 minutes under the following conditions: 0.2 M NaCl, 20 mM Tris HCl (pH 7.5), 0.25 mM EDTA and 5% glycerol. Each reaction was stopped by the addition of 30 l chloroform/iso-amyl alcohol (26:1) and 30 l stop dye (40% sucrose, 100 mM Tris.HCl (pH 7.5), 1 mM EDTA, 0.5 g/ml bromophenol blue), before being loaded on a 0.8% TAE gel run at 80V for 2 hours.
Topoisomerase II Relaxation assay 1 U of topo II was incubated with 0.5 g supercoiled plasmid DNA (pBR322) in a 30 l reaction at 37 °C for 30 minutes under the following conditions: 50 mM Tris HCl (pH 7.5),
125 mM NaCl, 10 mM MgCl 2, 5 mM DTT, 0.5 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA) and 1 mM ATP. Each reaction was stopped by the addition of 30 l chloroform/iso- amyl alcohol (26:1) and 30 l stop dye, before being loaded on a 0.8% TAE gel run at 80V for 3 hours.
Materials and Methods 215
Determination of the inhibitory activity towards Topo I and II Bands were visualized by ethidium staining for 10 minutes, destained and analyzed by gel documentation equipment (GeneGenius, Syngene, Cambridge, UK). Inhibition levels were obtained with gel scanning software (GeneTools, Syngene, Cambridge, UK) and statistical analysis was carried out using SigmaPlot (version 11.0). The data was plotted, curves fitted using SigmaPlot and the IC 50 values were calculated from the equation. The level of inhibition of relaxation was determined by measuring the amount of supercoiled plasmid as a percentage of the negative control (i.e.: no enzyme).
5.2.3.10 Alkaline comet assay
The comet assay or single cell gel electrophoresis (SCGE) assay is a classical method that can be used for detecting DNA damage at the level of individual cells. The comet assay was performed basically as described by Singh et al. 1988. The base slides with 1% normal melting point (NMP) agarose (Roth, Kalsruche) were prepared some days or weeks before the experiment by adding 1 ml of 1% NMP agarose onto frosted microscope slides. For performing the experiment, 100 µl of 0.7% NMP agarose was pipetted on the slide, covered with a cover slip and put on a flat surface on ice in the refrigerator at 4°C for 1 h. After treatment of MIA PaCa-2 cells with the diterpenes, cells were trypsinized, pelleted, and resuspended in 100 µl of 0.5% low melting point (LMP) agarose at 37 - 40°C and gently mixed. This mixture was immediately pipetted onto the previously pre-coated slide, covered with a coverslip and placed at 4°C to allow agarose solidify (1-2h). Slides were immersed in freshly prepared ice-cold lysis buffer (2.5 M NaCl, 0.1 M Na 2EDTA, 0.2 M NaOH, 1% Triton X-100, pH > 10) overnight. The slides were placed side by side in a gel electrophoresis tank, submerged with freshly made electrophoresis buffer (1 mM Na 2EDTA, 0.3 M NaOH, pH > 13) and incubated for 30 min to allow DNA unwinding prior to electrophoresis at 25 V/ 300 mA for 25 min. All steps after exposure were performed under dimmed light at 4°C. After electrophoresis slides were washed with neutralization solution composed of 0.4 M Tris-HCl pH 7.5 for 10 min, followed by washing with ultra pure water (2x 5 min) and air drying. Finally, nuclear DNA was stained with 10 µg/ml ethidium bromide and analyzed with a fluorescent microscope at 40x magnification. For each dose, 100 cells per slide were scored and recorded for analysis. Images were acquired and analyzed using the Kinetic Imaging 216 Materials and Methods
Komet 5.5 software. Comet length, percentage of DNA in the tail, and comet tail moment were used as parameters to evaluate the extension of DNA damage.
5.2.3.11 FACS and Cell cycle distribution analysis
Cell cycle analysis was carried out by flow cytometry according to Lamy & Mersch- Sundermann 2009. Fluorescence Activated Cell Sorter - FACS, or only flow cytometry is a technology that simultaneously measures multiple physical characteristics of single cells. The properties measured include particle relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to- electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence. In order to analyze a particular cell population, cells are labeled with fluorescent molecules, for example propidium iodide. Labeled cells flow through the illumination volume, and are irradiated by a laser, that then causes fluorescence emission. The emitted light is measured and recorded as a single event. The optics in modern flow cytometry instruments allow the simultaneous detection of fluorescence at four different wavelengths, plus the light diffraction at an angle of 0° and 90°. Consequently, up to four antigens can be simultaneously detected. In addition, the diffraction at 0° and 90° is proportional to cell size and granularity, respectively. These two parameters provide further information for cell subpopulation assignment. Once a cell sample is measured, the results can be analyzed with versatile software that allows the selection of a single cell type, positive for a particular cell population labeled with fluorescent molecules (gating), and perform further analyses only for the events that fall within the gated region. As a rule, 10000 events within the gate are counted. This permits the statistical analysis of an expressive cell population. Cancer progression has been suggested to involve the loss of cell cycle checkpoint controls that regulate the passage through the cell cycle. In parallel with the cytotoxic studies, cell cycle analysis was performed as previously described by Lamy & Mersch-Sundermann 2009. In brief, MIA PaCa-2 cells (2.5 x10 6 cells) were incubated with test substances for 24h. After treatment cells were harvested with trypsin-EDTA 0.25% and centrifuged (1200 rpm, 4°C, 5 min). Following washing steps with ice-cold PBS (2x) cells were fixed in cold 70% EtOH overnight (can be stored up to one week). After fixation, cells were washed again in ice-cold Materials and Methods 217
PBS (2x); pelleted by centrifugation (1200 rpm, 4°C, 5 min) and resuspended in 1 ml RNase solution with PI (RNase 10 µg/ml and PI 40 µg/ml, Sigma). DNA content of cells was measured, after 30 min incubation at 37°C, by a FACScan Cytometer (Beckton Dickinson, San Jose, CA) in the FL2 channel (575 nm). Percentage of cell cycle distribution in the G0/G1, S and G2/M phases were determined using the MODFIT software (Becton Dickinson). Ten thousand events were analyzed for each sample. Statistic analysis was done by comparing percentage of cells in each cell cycle stage from control cells and exposed cultures of 3 independent experiments with Student’s t-test.
5.2.3.12 Protein kinase assay
Regulation and maintenance of the cell cycle progression is a crucial process for cell survival. Two key classes of regulatory molecules, cyclins and cyclin dependent kinases (CDKs), determine a cell progress through the cell cycle. The assays on protein kinases were carried out by ProQinase® Tolls & Tests (Freiburg, Germany) according to the internal standard operating procedure (SOP). The test compounds were tested at 12 final assay concentrations in the range from 1 x 10 -4 M to 3 x 10 -9 M. A radiometric protein kinase assay (33 PanQinase ® Activity Assay) was used for measuring the kinase activity of the 9 protein kinases. All kinase assays were performed in 96-well FlashPlates TM from Perkin Elmer (Boston, MA, USA) in a 50 l reaction volume. The reaction cocktail was pipetted in 4 steps in the following order:
• 10 l of non-radioactive ATP solution (in water)
• 25 l of assay buffer /[ γ- 33 P]-ATP mixture
• 5 l of test sample in 10% DMSO
• 10 l of enzyme/substrate mixture
The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl 2 , 3 mM 5 MnCl 2, 3 M Na-orthovanadate, 1.2 mM DTT, 1 M ATP/[ γ- 33 P]-ATP (approx. 8 x 10 cpm per well), protein kinase (variable amounts; see Table 5.16) and substrate (variable amounts; see Table 5.16) 218 Materials and Methods
Table 5.16 - Amounts of each kinase and substrate used in the protein kinase assay. Conc. Conc. No Kinase Lot. Substrate Lot. (ng/50µl) (ng/50 µl) 1 CDK1/CycB1 25 25 RBER-CHKtide 24 2 2 CDK2/CycE 9 10 RBER-CHKtide 9 1 3 CDK3/CycE 1 10 RBER-CHKtide 24 1 4 CDK4/CycD3 1 10 RBER-CHKtide 24 1 5 CDK6/CycD1 4 200 RBER-CHKtide 24 2 6 CDK7/CycH/MAT1 2 25 RBER-CHKtide 24 2 7 CDK8/CycC 2 50 RBER-IRStide 1 1 8 CDK9/CycT 4 15 RBER-CHKtide 24 1 9 PCTAIRE1 4 400 RBER-CHKtide 24 2
Recombinant Protein Kinases All protein kinases were expressed in Sf9 insect cells or in E. coli as human recombinant GST-fusion proteins or His-tagged proteins. Kinases were purified by affinity chromatography using either GSH-agarose (Sigma) or Ni-NTH-agarose (Qiagen). The purity of each kinase was checked by SDS-PAGE/silver staining and the identity of each kinase was verified by mass spectroscopy. The reaction cocktails were incubated at 30°C for 60 minutes.
The reaction was stopped with 50 l of 2% (v/v) H 3PO 4, plates were aspirated and washed two times with 200 l 0.9% (w/v) NaCl. Incorporation of 33 Pi (counting of “cpm”) was determined with a microplate scintillation counter (Microbeta, Wallac). All assays were performed with a Beckman Coulter Biomek 2000/SL robotic system.
Evaluation of Raw Data For each kinase, the median value of the cpm of three wells with complete reaction cocktails, but without kinase, was defined as "low control" (n=3). This value reflects unspecific binding of radioactivity to the plate in the absence of protein kinase but in the presence of the substrate. Additionally, for each kinase the median value of the cpm of three other wells with the complete reaction cocktail, but without any compound, was taken as the "high control", i.e. full activity in the absence of any inhibitor (n=3). The difference between high and low control was taken as 100% activity for each kinase. As part of the data evaluation the low control value of each kinase was subtracted from the high control value as Materials and Methods 219 well as from their corresponding "compound values". The residual activity (in %) for each compound was calculated by using the following formula: Res. Activity (%) = 100 X [(cpm of compound – low control)/(high control – low control)] Since 10 distinct concentrations of each test compound were tested against each kinase, the evaluation of the raw data resulted in 10 values for residual activities per kinase. Based on each 10 corresponding residual activities, IC 50 values were calculated using Prism 5.03 for Windows (Graphpad, San Diego, California, USA). The mathematical model used was "Sigmoidal response (variable slope)“ with parameters "top“ fixed at 100% and "bottom“ at 0%.
5.2.3.13 p38 ααα MAPK assay
p38 α MAP kinase assay determine the effect of a potential inhibitor candidate using the amount of the phosphorylated kinase substrate ATF-2 as indicative of the enzyme activity. The n-hexane extract of P. longipes as well as the main isolated compounds were tested at 100 µg/ml and 0.1, 1, 10 and 100 µM concentrations, respectively, according to the enzyme-linked immunossorbent assay (ELISA) described by Laufer and co-workers (Laufer et al. 2005; Goettert et al. 2010). The reference compound SB203580 was also tested at 0.01, 0.1, 1 and 10 µM concentrations. A 100 µM ATP concentration was used and the primary antibody (phospho-ATF-2(Thr69/71)-antibody) was detected by a second antibody (anti-rabbit IgGAP- antibody) which dephosphorylates 4-nitrophenylphosphate allowing the 4-nitrophenyl to be detected photometrically at a wavelength of 405 nm. This ELISA-based p38 α assay consists of the following steps:
1. The microtiter well plates were coated with 50 µL/well of the kinase substrate ATF-2 (10 µg/ml in TBS), incubated for 90 min at 37 °C and washed 3 times with bidistilled water;
2. The blocking buffer (BB) containing 0.25% BSA, 0.02% sodium azide and 0.05% tween 20 in TBS was added and incubated for 30 min at room temperature;
3. After washing 3x with bidistilled water, 50 µl of the test solutions containing 12 ng/well of p38 MAPK were diluted in the kinase buffer (50 mM tris, pH 7.5, 10 mM MgCl 2, 10 mM
β-glycerophosphate, 100 µg/ml BSA, 1 mM dithiothreitol, 0.1 mM Na 3VO 4, 100 µM rATP) 220 Materials and Methods with or without the compounds. The compounds were dissolved in DMSO. The plates were incubated for 1 h at 37 °C.
4. After subsequent washing, plates were blocked again with BB for 15 min followed by a fourth washing step. Wells were filled with 50 µl of the first AB (1:500 in BB) and incubated for 1 h at 37 °C followed by washing and consecutive incubation with 50 µl of the second AB (alkaline phosphatase conjugated) (1:4000 in BB).
5. After a final washing step 100 µl of 4-NPP were added in each well and the color development was measured after 1.5 - 2h with a microplate reader at 405 nm wavelength.
As negative control, a solution of kinase buffer and ATP without p38 α and inhibitor was used. The non-specific binding value obtained from this solution was subtracted from all samples and positive control to calculate the inhibition rate.
The relative inhibition is calculated by the following equation:
Relative Inhibition [%] = [100 – (OD Comp / OD Stim )] x 100
OD Comp : mean of the optical density in 3 wells of the corresponding compounds.
OD Stim : mean of the optical density in the non-inhibited stimulation controls.
The IC 50 value was defined as concentration which inhibits 50% of the enzyme activity. It was graphically constructed by interpolation of the semi-logarithmic plot of the inhibition (%) against the inhibitor concentration (log c).
5.2.4. Docking studies
The docking approach of diterpenes and camptothecin to topoisomerase I (Topo I) was performed in cooperation with Prof. Dr. Stefan Günther (Pharm. Bioinformatics, Uni Freiburg, Germany). The co-crystallized structure of topoisomerase I, DNA and camptothecin (PDB: 1T8I) was applied as a template structure for a docking approach using the software package Glide (Schrödinger, Inc.). The diterpenes were docked into the predetermined binding pocket which is characterized by camptothecin using the Induced Fit protocol of Glide. Alternative docking modes were evaluated using the DNA-unbound form of Topo I (PDB: 1A36). Potential binding pockets were determined by FTMAP algorithm. Based on Materials and Methods 221
Fourier domain correlation techniques the algorithm identified five different pockets located at the surface of the DNA-interaction site. The diterpenes were docked into the proposed binding pockets using the Glide QM-Polarized Ligand Docking protocol.
5.2.5. Statistical analysis
Statistical evaluation was carried out with different programs and software’s as Origin Scientific Graphing and Analysis Software, version 7.0, GraphPad (GraphPad Prism 5) computer program or with Microsoft Office Excel 2007. The IC 50 values with 95% confidence interval were obtained by non-linear regression using GraphPad. Data are in general expressed as the mean ± S.E.M. or S.D.. Significant differences between the treated groups and the control were determined by the Student’s t-test, at different levels.
222 References
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List of figures 237
7. List of figures
Figure 1.1 - Peltodon longipes subshrub or herb...... 6
Figure 1.2 - Flowers and roots of Peltodon longipes ...... 7
Figure 1.3 - Worldwide distribution of Peltodon longipes ...... 7
Figure 1.4 - Isoprenoid biosynthesis via the acetate-mevalonate pathway (adapted from Chinou 2005)...... 9
Figure 1.5 - Classification of diterpenes according to the number of ring systems present (Hoffmann 2006)...... 10
Figure 2.1 - Fluorescent microscope images to evaluate wound healing in vitro in the scratch assay using a confluent monolayer of 3T3 fibroblasts. Cell migration and proliferation into the wound was observed in response to an artificial injury. A single representative area is shown immediately after the wounding (A), a control group (B), after treatment with 2 ng/ml PDGF (C), and after treatment of 10 µg/ml of an n-hexane extract of Calendula officinalis (D) after 12h of incubation...... 26
Figure 2.2 - Effect of 0.5, 1, 2, 4 and 15 ng/ml of PDGF after 12h of incubation (37 ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS on confluent monolayer cultures of fibroblasts. Data are expressed as percentage of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments...... 26
Figure 2.3 - Effect of 2 ng/ml of PDGF after 8, 10, 12 and 14h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS on confluent monolayer cultures of 3T3 fibroblasts. Data are expressed as percent of cell numbers in the wounded area compared to the control. Bars represent the mean ± S.E.M. of three experiments...... 27
Figure 2.4 - Effect of preparations from Calendula officinalis , Matricaria recutita and Hypericum perforatum oil on the migratory and proliferative activities of fibroblasts in the scratch assay after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS...... 28
Figure 2.5 - Effect of PDGF, n-hexane extract of Calendula officinalis and triterpene esters on the migratory activities of fibroblasts in the scratch assay in the presence of 5 µg/ml of antimitotic mitomycin C after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS...... 29
Figure 2.6 - Effect of the triterpene esters faradiol myristate and faradiol palmitate on the migratory and proliferative activities of fibroblasts in the scratch assay after 12h of incubation (37ºC, 5% CO 2) in DMEM medium supplemented with 10% FBS...... 30
Figure 2.7 - HPLC chromatogram of the n-hexane extract of Peltodon longipes with the respective identified diterpene by LC-NMR analysis...... 34
Figure 2.8 - Chemical structure of 7 α-acetoxyroyleanone...... 35
Figure 2.9 - EI-MS spectrum of 7 α-acetoxyroyleanone (1)...... 38
13 Figure 2.10 - C-NMR spectrum of 7 α-acetoxyroyleanone (1) (100 MHz, CDCl 3)...... 39 238 List of figures
1 Figure 2.11 - H-NMR spectrum of 7 α-acetoxyroyleanone (1) (400 MHz, CDCl 3)...... 40
Figure 2.12 - HMBC spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3)...... 41
Figure 2.13 - H;H-COSY spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3)...... 42
Figure 2.14 - HSQC spectrum of 7 α-acetoxyroyleanone (1) (CDCl 3)...... 43
Figure 2.15 - Chemical structure of 7 α-hydroxyroyleanone (2)...... 45
Figure 2.16 - EI-MS spectrum of 7 α-hydroxyroyleanone (2)...... 46
13 Figure 2.17 - C-NMR spectrum of 7 α-hydroxyroyleanone (2) (100 MHz, CDCl 3)...... 47
1 Figure 2.18 - H-NMR spectrum of 7 α-hydroxyroyleanone (2) (400 MHz, CDCl 3)...... 48
Figure 2.19 - HSQC spectrum of 7 α-hydroxyroyleanone (2) (CDCl 3)...... 49
Figure 2.20 - HMBC spectrum of 7 α-hydroxyroyleanone (2) (CDCl 3)...... 50
Figure 2.21 - H;H-COSY spectrum of 7 α-hydroxyroyleanone (2) (CDCl 3)...... 51
Figure 2.22 - Chemical structure of royleanone (3)...... 53
Figure 2.23 - EI-MS spectrum of royleanone (3)...... 54
13 Figure 2.24 - C-NMR spectrum of royleanone (3) (100 MHz, CDCl 3)...... 55
1 Figure 2.25 - H-NMR spectrum of royleanone (3) (400 MHz, CDCl 3)...... 56
Figure 2.26 - HSQC spectrum of royleanone (3) (CDCl3)...... 57
Figure 2.27 - HMBC spectrum of royleanone (3) (CDCl3)...... 58
Figure 2.28 - H;H-COSY spectrum of royleanone (3) (CDCl 3)...... 59
Figure 2.29 - Chemical structure of 7-ketoroyleanone (4)...... 61
Figure 2.30 - EI-MS spectrum of 7-ketoroyleanone (4)...... 62
13 Figure 2.31 - C-NMR spectrum of 7-ketoroyleanone (4) (100 MHz, CDCl 3)...... 63
1 Figure 2.32 - H-NMR spectrum of 7-ketoroyleanone (4) (400 MHz, CDCl 3)...... 64
Figure 2.33 - HSQC spectrum of 7-ketoroyleanone (4) (CDCl 3)...... 65
Figure 2.34 - HMBC spectrum of 7-ketoroyleanone (4) (CDCl 3)...... 66
Figure 2.35 - Chemical structure of 7 α-ethoxyroyleanone (5)...... 68
Figure 2.36 - EI-MS spectrum of 7 α-ethoxyroyleanone (5)...... 69
13 Figure 2.37 - C-NMR spectrum of 7 α-ethoxyroyleanone (5) (100 MHz, CDCl 3)...... 70
1 Figure 2.38 - H-NMR spectrum of 7 α-ethoxyroyleanone (5) (400 MHz, CDCl 3)...... 71 List of figures 239
Figure 2.39 - HSQC spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3)...... 72
Figure 2.40 - HMBC spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3)...... 73
Figure 2.41 - H;H-COSY spectrum of 7 α-ethoxyroyleanone (5) (CDCl 3)...... 74
Figure 2.42 - Chemical structure of iguestol (6)...... 76
Figure 2.43 - EI-MS spectrum of iguestol (6)...... 77
1 Figure 2.44 - H-NMR spectrum of iguestol (6) (400 MHz, CDCl 3)...... 78
13 Figure 2.45 - C-NMR spectrum of iguestol (6) (100 MHz, CDCl 3)...... 79
Figure 2.46 - HSQC spectrum of iguestol (6) (CDCl 3)...... 80
Figure 2.47 - HMBC spectrum of iguestol (6) (CDCl 3)...... 81
Figure 2.48 - H;H-COSY spectrum of iguestol (6) (CDCl 3)...... 82
Figure 2.49 - Chemical structure of deoxyneocryptotanshinone (7)...... 84
Figure 2.50 - EI-MS spectrum of deoxyneocryptotanshinone (7)...... 85
13 Figure 2.51 - C-NMR spectrum of deoxyneocryptotanshinone (7) (100 MHz, CDCl 3)...... 86
1 Figure 2.52 - H-NMR spectrum of deoxyneocryptotanshinone (7) (400 MHz, CDCl 3)...... 87
Figure 2.53 - HMBC spectrum of deoxyneocryptotanshinone (7) (CDCl 3)...... 88
Figure 2.54 - H;H-COSY spectrum of deoxyneocryptotanshinone (7) (CDCl 3)...... 89
Figure 2.55 - HSQC spectrum of deoxyneocryptotanshinone (7) (CDCl 3)...... 90
Figure 2.56 - Chemical structure of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8)...... 92
Figure 2.57 - EI-MS spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8)...... 93
Figure 2.58 - 13 C-NMR spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (100 MHz, CDCl 3)...... 94
Figure 2.59 - 1H-NMR spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (400 MHz, CDCl 3)...... 95
Figure 2.60 - HMBC spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3)...... 96
Figure 2.61 - HSQC spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3)...... 97
Figure 2.62 - H;H-COSY spectrum of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) (CDCl 3)...... 98
Figure 2.63 - Chemical structure of inuroyleanol (9)...... 100
Figure 2.64 - EI-MS spectrum of inuroyleanol (9)...... 101
13 Figure 2.65 - C-NMR spectrum of inuroyleanol (9) (100 MHz, CDCl 3)...... 102 240 List of figures
1 Figure 2.66 - H-NMR spectrum of inuroyleanol (9) (400 MHz, CDCl 3)...... 103
Figure 2.67 - HMBC spectrum of inuroyleanol (9) (CDCl 3)...... 104
Figure 2.68 - HSQC spectrum of inuroyleanol (9) (CDCl 3)...... 105
Figure 2.69 - H;H-COSY spectrum of inuroyleanol (9) (CDCl 3)...... 106
Figure 2.70 - Chemical structure of sugiol (10)...... 108
Figure 2.71 - EI-MS spectrum of sugiol (10)...... 109
13 Figure 2.72 - C-NMR spectrum of sugiol (10) (100 MHz, CDCl 3)...... 110
1 Figure 2.73 - H-NMR spectrum of sugiol (10) (400 MHz, CDCl 3)...... 111
Figure 2.74 - HMBC spectrum of sugiol (10) (CDCl 3)...... 112
Figure 2.75 - HSQC spectrum of sugiol (10) (CDCl 3)...... 113
Figure 2.76 - H;H-COSY spectrum of sugiol (10) (CDCl 3)...... 114
Figure 2.77 - Chemical structure of cryptojaponol (11)...... 116
Figure 2.78 - EI-MS spectrum of cryptojaponol (11)...... 117
13 Figure 2.79 - C-NMR spectrum of cryptojaponol (11) (100 MHz, CDCl3)...... 118
1 Figure 2.80 - H-NMR spectrum of cryptojaponol (11) (400 MHz, CDCl3)...... 119
Figure 2.81 - HMBC spectrum of cryptojaponol (11) (CDCl 3)...... 120
Figure 2.82 - HSQC spectrum of cryptojaponol (11) (CDCl 3)...... 121
Figure 2.83 - H;H-COSY spectrum of cryptojaponol (11) (CDCl 3)...... 122
Figure 2.84 - Chemical structure of orthosiphonol (12)...... 124
Figure 2.85 - EI-MS spectrum of orthosiphonol (12)...... 125
13 Figure 2.86 - C-NMR spectrum of orthosiphonol (12) (100 MHz, CDCl3)...... 126
1 Figure 2.87 - H-NMR spectrum of orthosiphonol (12) (400 MHz, CDCl3)...... 127
Figure 2.88 - HMBC spectrum of orthosiphonol (12) (CDCl 3)...... 128
Figure 2.89 - HSQC spectrum of orthosiphonol (12) (CDCl 3)...... 129
Figure 2.90 - H;H-COSY spectrum of orthosiphonol (12) (CDCl 3)...... 130
Figure 2.91 - HPLC chromatogram of the n-hexane extract from Peltodon longipes prepared from the roots (injection: 10 µl = 5 µg extract), concentrations of the main compound: 1: 24.3%. Further HPLC conditions are given in the Experimental Section 5.2.1...... 132
Figure 2.92 - Cell death (%) after different exposition time in MIA PaCa-2 cells with 0.5 to 16 µM of 7α-acetoxyroyleanone (1) evaluated by MTT assay...... 133 List of figures 241
Figure 2.93 - Chemical structures of the diterpenes isolated from Peltodon longipes (1 - 12), Salvia miltiorrhiza (13 - 19) and Salvia sahendica (20 and 21)...... 135
Figure 2.94 - Cytotoxic activity of 7 α-acetoxyroyleanone (1) evaluated by FACS analysis after DNA staining with PI. MIA PaCa-2 cells were treated with the same conditions as in the MTT assay...... 136
Figure 2.95 - Comparative reaction rates of diterpenes 1 - 4 and 10 at various concentrations with NBP at 37°C (for detailed information see section 5.2.3.8). p-Benzoquinone was used as positive control. Data are mean (± SD) of three independent experiments ...... 141
Figure 2.96 - Proposed chemical reaction of the diterpene 1 with NBP...... 142
Figure 2.97 - Reaction rates of diterpene 1 at various concentrations with NBP at 37°C (for detailed information see section 5.2.3.8) in the presence ( •) or absence ( x) of 10 mM concentration of DTT. Data are mean (± SD) of two independent experiments...... 143
Figure 2.98 - Concentration dependent effects of diterpenes 1 - 4 and 10 on topoisomerase I mediated relaxation of pBR322 DNA...... 144
Figure 2.99 - Concentration dependent effect of diterpenes 4 and 10 on topoisomerase II mediated relaxation of pBR322 DNA...... 146
Figure 2.100 - Docking approach of camptothecin and diterpenes to topoisomerase I...... 148
Figure 2.101 - Effect of diterpene 1 (7 α-acetoxyroyleanone) (A), 4 (7 ketoroyleanone) (B) and 10 (sugiol) (C) on cell cycle progression...... 150
Figure 2.102 - Study of the effect on caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 10, 16 and 24h with the indicated concentrations of the n-hexane extract of P. longipes. Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments...... 151
Figure 2.103 - Effect of caspase-3/7 using the fluorogenic caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 10, 16 and 24h with the indicated concentrations of 7 α-acetoxyroyleanone (1). Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments...... 152
Figure 2.104 - Effect on caspase-3/7 using the fluorogenic caspase-3/7 assay (depicted as relative fluorescence units/ RFU) in MIA PaCa-2 cells treated for 24h with the indicated concentrations of horminone (2), royleanone (3), 7-ketoroyleanone (4) and sugiol (10). Actinomycin D (ActD) was used as positive control. Error bars represent the mean ± S.E.M. of two independent experiments...... 153
Figure 2.105 - Effects on apoptosis-associated DNA fragmentation using the cell death detection ELISA plus Kit (depicted as enrichment factor) after 16h exposure in MIA PaCa-2 cells with the indicated concentrations of the n-hexane extract of Peltodon longipes , 7 α-acetoxyroyleanone (1), horminone (2) and royleanone (3). Actinomycin D (ActD) was used as positive control...... 154
Figure 2.106 - Apoptotic cells measured as alteration in the “sub-G1” DNA content after 24h treatment with different concentrations of diterpenes 1 - 4 and 10. Camptothecin (CPT) was used as positive control...... 155
Figure 2.107 - Effect of 7 α-acetoxyroyleanone (1) (A) and sugiol (10) (B) on DNA damage in MIA PaCa-2 cells evaluated by the alkaline comet assay after 1h treatment with the indicated concentrations...... 158 242 List of figures
Figure 2.108 - Repair of DNA damage measured by the alkaline comet assay. MIA PaCa-2 cells were exposed for 1h with camptothecin (5 µM), sugiol [(10) 40 µM] and 7 α-acetoxyroyleanone [(1) 4 µM] and the DNA repair potential was evaluated after 12 and 24h...... 159
Figure 2.109 - Determination of the apoptotic effects involved in the observed cytotoxicity of diterpene 1 (7 α-acetoxyroyleanone) and the n-hexane extract of P. longipes in MIA PaCa-2 cells after 24h exposure using the pan-caspase inhibitor (Q-VD-OPH)...... 160
Figure 2.110 - Determination of the proportion of apoptotic MV-3 cells within the killed cells after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7 α- acetoxyroyleanone (1), studied with and without the pan-caspase inhibitor (Q-VD-OPH). Error bars represent the mean ± S.E.M. of two independent experiments...... 161
Figure 2.111 - Influence of ROS scavenger BHA on cell death in MIA PaCa-2 after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7 α-acetoxyroyleanone (1). 162
Figure 2.112 - Influence of ROS scavenger BHA on cell death of MV-3 after 24h exposure with 1 and 2 µg/ml of the n-hexane extract of P. longipes and 4 and 8 µM of 7 α-acetoxyroyleanone (1). Error bars represent the mean ± S.E.M. of two independent experiments...... 162
Figure 2.113 - ROS accumulation after MIA PaCa-2 cell exposed to the extract of P. longipes , 7 α- acetoxyroyleanone (1), 7-ketoroyleanone (4) and sugiol (10) for 24h with the indicated concentrations. ROS was measured using the dichlorofluorescein fluorescence assay and referred to untreated control...... 163
Figure 3.1 - A - Proposed mechanism by which topoisomerase I controls the topological state of DNA at normal conditions. B - inhibitors of the topoisomerase-DNA cleavage complex, “interfacial inhibitors”. C and D - topoisomerase I catalytic inhibitors (Adapted from Wu et al. 2010)...... 174
Figure 5.1 - Extraction and isolation scheme of the diterpenes from the n-hexane extract of Peltodon longipes ...... 199
Figure 5.2 - TLC of the main fractions (F1 to F15) obtained during fractionation by OC of the filtrated part from the n-hexane extract of P. longipes . TLC silica gel plates were eluted with EtOAc: n-hexane (35:75) and detection was performed with anisaldehyde-sulphuric reagent...... 200
Figure 5.3 - TLC of the main fractions (RF1 to RF4) obtained during fractionation by OC of the residue part from the n-hexane extract of P. longipes . TLC silica gel plate was eluted with petroleum ether:EtOAc (20:3) and detection was done with anisaldehyde-sulphuric reagent...... 203
Figure 5.4 - MTT reaction...... 208
Figure 5.5 - Explanation of the gels. 1 UI of topo I and/or II enzyme were needed to relax the supercoiled plasmid DNA (pBR322)...... 214
List of tables 243
8. List of tables
Table 2.1 - Effects of Brazilian plant extracts at 10 µg/ml concentration on cellular migration and proliferation evaluated in the scratch assay...... 31
Table 2.2 - Results from the elastase assay using the n-hexane and the ethanolic extracts of twelve Brazilian medicinal plants...... 33
Table 2.3 - NMR data of 7 α-acetoxyroyleanone (1) compared to the NMR data from the literature*. 44
Table 2.4 - NMR data of 7 α-hydroxyroyleanone (2) compared to the NMR from the literature*...... 52
Table 2.5 - NMR data of royleanone (3) compared to the NMR from the literature*...... 60
Table 2.6 - NMR data of 7-ketoroyleanone (4) compared to the NMR from the literature*...... 67
Table 2.7 - NMR data 7 α-ethoxyroyleanone (5) compared to the NMR from the literature*...... 75
Table 2.8 - NMR data iguestol (6) compared to the NMR data from the literature*...... 83
Table 2.9 - NMR data of deoxyneocryptotanshinone (7)...... 91
Table 2.10 - NMR data of 12-hydroxy-11-metoxyabieta-8,11,13-trien-7-one (8) compared to the NMR from the literature*...... 99
Table 2.11 - NMR data of inuroyleanol (9) compared to the NMR data from the literature*...... 107
Table 2.12- NMR data of sugiol (10) compared to the NMR data from the literature (*)(**)...... 115
Table 2.13 - NMR data of cryptojaponol (11) compared to the NMR data from the literature*...... 123
Table 2.14 - NMR data of orthosiphonol (12) compared to the NMR data from the literature*...... 131
Table 2.15 - Cytotoxic activity of the diterpenes 1 - 12 isolated from Peltodon longipes and 13 - 21 from Salvia species in MIA PaCa-2, a human pancreatic carcinoma cell line and MV-3, a human melanoma cancer cell line using the MTT assay. Data are presented as IC 50 values and 95% confidence interval from three independent experiments. Camptothecin was used as positive control...... 134
Table 2.16 - Effect of 7 α-acetoxyroyleanone (1) at a 10 µM concentration on NCI 60 human solid tumor cells represented as growth percentage...... 138
Table 2.17 - Effect of royleanone (3) at a 10 µM concentration on NCI 60 human solid tumor cells represented as growth percentage...... 139
Table 2.18 - Effect of 7 α-acetoxyroyleanone (1) against the 60 cell line panel at five concentrations. Results are shown as GI 50 (growth inhibitory), TGI 50 (cytostatic effect) and LC 50 (cytotoxic effect). 140
Table 2.19 - Inhibitory effects of diterpenes 1 - 4 and 10 on human DNA topoisomerase I and II (IC 50 inhibition rate of relaxation). IC 50 values (µM) and the standard error of the mean ± S.E.M. for n = 2 are given...... 145 244 List of tables
Table 2.20 - Virtual binding affinity to topoisomerase I determined by FTMAP algorithm for diterpenes 1 - 4 and 10 with the respective inhibitory effects on DNA topoisomerase I (IC 50 inhibition rate of relaxation)...... 147
Table 2.21 - Effect of 7 α-acetoxyroyleanone and sugiol on nine protein kinase of the cell cycle 33 ® evaluated in the radiometric protein kinase assay ( PanQinase Activity Assay) IC 50 values are given in Molar (M)...... 156
Table 2.22 - Inhibitory activity of the n-hexane extract of P. longipes and the respective isolated diterpenes on p38 α MAPK. IC 50 values are given in µM and represent the mean ± S.E.M. for n=3. SB203580 was used as reference substance and showed an IC 50 of 0.017 µM (± 0.002)...... 164
Table 5.1 - List of used chemicals...... 185
Table 5.2 - Cytokines, antibodies, enzymes and kits used in this work...... 187
Table 5.3 - List of used consumables...... 188
Table 5.4 - List of used equipment...... 190
Table 5.5 - Selected plants used in the screening phase...... 192
Table 5.6 – Amounts of each fraction obtained by fractionating the n-hexane extract (filtrate part) of Peltodon longipes ...... 199
Table 5.7 - Amounts of each subfraction obtained after chromatographic separation of F1...... 201
Table 5.8 - Amounts of each subfraction obtained after chromatographic separation of F8...... 201
Table 5.9 - Amounts of each subfraction obtained after chromatographic separation of F11...... 201
Table 5.10 - Amounts of each subfraction obtained after chromatographic separation of F13...... 202
Table 5.11 - Amounts of each subfraction obtained after chromatographic separation of F14...... 202
Table 5.12 - Amount of each fraction obtained through fractionation of the n-hexane extract (residue part) form the roots of Peltodon longipes...... 203
Table 5.13 - Amounts of each subfraction obtained after chromatographic separation of RF2...... 204
Table 5.14 - Amounts of each subfraction obtained after chromatographic separation of RF2.SF4 combined with RF2.SF5...... 204
Table 5.15 - List of used cells and their respective culture medium composition and supplier...... 205
Table 5.16 - Amounts of each kinase and substrate used in the protein kinase assay...... 218
Table 9.1 - List of abbreviations...... 245
List of abbreviations 245
9. List of abbreviations
Table 9.1 - List of abbreviations. Abbreviation ActD Actinomycin D ACN Acetonitrile ATP Adenosine triphosphate BHA Butylated Hydroxyanisole br Broad BSA Bovine serum albumin CC Column chromatography CDKs Cyclin dependent kinase CDKI Cyclin dependent kinase inhibitor
CH 2Cl 2 Dichloromethane cm Centimeter Cyc Cyclin CPT Camptothecin d Doublet Da Dalton DAD Diode Array Detector DAPI 4´,6-diamino-2-phenylindole DMEM Dubelcco´s modified Eagle´s medium DMF Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DTT Dithiothreitol ECM Extracellular matrix EI-MS Electron Ionization Mass Spectrometry ELISA Enzyme-linked immunosorbent assay ESI-MS Electrospray Ionization Mass Spectrometry ETO Etoposide EtOAc Ethylacetate EtOH Ethanol FA Formic acid FACS Fluorescence Activated Cell Sorter FBS Fetal bovine serum FCS Fetal calf serum g Gram 246 List of abbreviations h Hour (s)
H2 O Water
H2DCFDA 2,7-dichloro-dihydrofluorescin diacetate HCOOH Formic acid HNE Human neutrophil elastase HPLC High pressure liquid chromatography Hz Hertz IL Interleukin J Coupling L Liter LMP Low melting point LPLC Low pressure liquid chromatography LPS Lipopolysaccharide MAPK Mitogen activated protein kinase MeOH Methanol MIA PaCa-2 Human pancreatic cancer cell line min Minute MKK MAPK kinase mRNA Messenger RNA MS Mass spectrometry 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium MTT bromide MV-3 Human melanoma cancer cell line NBP 4-(4-nitro - benzyl) pyridine NF-κB Nuclear factor-kappaB NMP Normal melting point NMR Nuclear Magnetic Resonance NP-40 Nonidet P-40 OC Open column chromatography OTM Olive tail moment PAF Platelet-activating factor PBS Phosphate-buffered saline PDGF Platelet-derived growth factor PH Partial hepatectomy PK Protein kinase PI Propidium Iodide qVD-OPh Q-Val-Asp O-Ph, non-O-methylated RFU Relative fluorescence unit ROS Reactive oxygen species RP Reversed phase rpm Rounds per minute List of abbreviations 247
RT Retention time SAR Structure activity relationship SCGE single cell gel electrophoresis SDS Dodecil sulfato de sodio SEM Standard error of the mean SD Standard Deviation TLC Thin layer chromatography TNF α Tumor necrosis factor α TOPO Topoisomerase UV Ultraviolet
248 Acknowledgment
10. Acknowledgment
This thesis was realized at the Department of Pharmaceutical Biology and Biotechnology at University of Freiburg under the supervision of Frau Prof. Dr. Irmgard Merfort, whom I am deepest grateful for her continuous support, encouragement, inexhaustible optimism, constructive suggestions and discussions during my research. Finally for the countless hours of attention she devoted throughout the course of my thesis and for her help finishing this work. I am thankful to Prof. Dr. Stefan Laufer from Eberhard-Karls-Universität of Tübingen for the motivation and support to my work and for adopting the position of second reviewer. My thank you also goes to Prof. Dr. Andreas Bechthold from the Department of Pharmaceutical Biology and Biotechnology at University of Freiburg for acting as a third examiner. Many thanks to Prof. Dr. Berta Heinzmann from Federal University of Santa Maria, Brazil, for the confidence and support. Thanks also for many hours expend collecting the plants and the preparation of the extracts. Thanks to the Forestry Engineer Prof. Dr. Solon J. Longhi and the Botanists Dr. Gilberto D. Zanetti from Federal University of Santa Maria, Brazil, for the identification of the plants. My special thanks to Prof. Dr. Mathias Hamburger and his group from the Department of Pharmaceutical Sciences, University of Basel, Switzerland, for providing the natural compounds from Calendula and from Salvia species; moreover, for the helpful contributions and suggestions in our publications. I am grateful to Prof. Dr. Renato Murillo from Universidad de Costa Rica, San José, Costa Rica, for his support and help in the interpretation of NMR spectra and for his friendship. My thanks also extend to Mr. Brecht from the Department of Pharmaceutical and Medicinal Chemistry, for recording the NMR spectra; Dr. Wörth and C. Warth from the Institute of Organic Chemistry for the recording the MS spectra; Prof. Dr. Stefan Günther from the Pharmaceutical Bioinformatics lab for the molecular docking studies. All from University of Freiburg. A sincere thank you goes to the “master of comets” Robert Burzan and Mathias Könczöl from the Department of Environmental Health Science, University Freiburg, for the intensive lab work with the experiments on DNA damage, the fruitful discussions and the friendship. My thank you to Dr. Evelyn Lamy and her technical assistant Bärbel Junge from the Acknowledgment 249
Department of Environmental Health Science, University Freiburg for the cell cycle analysis and scientific discussions. Thanks to Prof. Dr. Meyer and Thomas Wilmes from the Institute of Experimental Pharmacology and Toxicology, University Freiburg, for helpful advices and support for establishing the scratch assay. Thanks to the Brazilian colleagues Fabiana Geller for the motivation and support; and Marcia Goettert for carrying out the p38 assays from Eberhard-Karls-Universität of Tübingen. In addition, of course, my special thanks to all friends and colleagues at the Department of Pharmaceutical Biology and Biotechnology. I am deepest grateful to all the actual and former members of the Merfort group for the excellent atmosphere, cordiality and colleagueship which makes the working days much more easy and pleasant. To my German brother Christoph Jäger, for his help and support at any time in the lab work and the great time expend together outside of the university, doing mountain bike tours and getting into the german culture of drinking beer. Kathrin Schmich for the scientific discussions about “significant” results and help with phone calls and orders. Katrin Nauman for sharing experiences with the scratch assay. Sandra Ebeling and Anna Lutz for all help and critical suggestions. Andrea Hrenn for teaching me the elastase assay. Cleber Schimdt for introducing me to the working group and to the majority of lab techniques. Titus Sparna for the friendship and together with Christoph for being my colleagues during lunch at delicious Mensa. Agnes Millet for the ecological conscience. Bettina Siedle for supporting and taking care of the students. Barbara Schuler for the help with phytochemical and analytical procedures, and for fulfil the lab with the accessories and supplies necessary to our work. Torsten Lingott for the great time sharing the office and important discussions on Brazilian football and German beers and Hitesh Patel for the arguments about spice food. Thank you all for the great time we share inside and as well outside of the lab during this time. Additionally, my thanks to the Government of Baden-Württemberg (Zukunftsoffensive IV “Innovation und Exzellenz”) for the financial support to the project. Finally, I wish to expresses my heartfelt gratitude to my family and Angelita for their love, continuous encouragement and support, extra patience and motivation in reaching my aims and to make this possible.
Marcio Fronza 250 Curriculum vitae
11. Curriculum vitae
Personal Details Name: Marcio Fronza Sex: Male Date of Birth: 08 th of January 1978 Nationality: Brazilian Parents: Domingos Fronza Neto and Carmelina De Carli Fronza E-mail: [email protected]
Education
2007 - Present PhD student, Department for Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg in Br., Germany Advisor: Prof. Dr. Irmgard Merfort Thesis entitled : Phytochemical investigation of the roots of Peltodon longipes and in vitro cytotoxic studies of abietane diterpenes
2004 - 2006 MSc in Pharmaceutical Science and Technology, Department of Industrial Pharmacy, Federal University of Santa Maria, Brazil Advisor: Prof. Dr. Sergio Dalmora Thesis entitled : Development and validation of methodologies for analysis of valdecoxib. Evaluation of the effects on hematological parameters
1997 - 2002 Graduation in Pharmacy and Biochemistry: Specialization in Industrial Pharmacy, Federal University of Santa Maria, Brazil
1993 - 1995 High School - Bento Gonçalves School, Tucunduva, RS - Brazil
Professional Experience
01/2004 - 06/2006 Supervisor - Center for Bioavailability and Pharmacokinetics Studies of Pharmaceutical Products, Federal University of Santa Maria, Santa Maria, RS, Brazil
05/2003 - 05/2005 Lecturer - Pharmaceutical Technology, Federal University of Santa Maria, Santa Maria, RS, Brazil
02/2002 - 05/2004 Analyst - Center for Tests and Analysis of Pharmaceutical Products, Federal University of Santa Maria, Santa Maria, RS, Brazil
06/2001 - 12/2001 Trainee - Pharmaceutical industry - Laboratório Químico Farmacêutico Bergamo, São Paulo, SP, Brazil
01/2000 - 03/2000 Trainee - Semi-industrial Laboratory and Hospital Pharmacy - Hospital Dr. Bartholomeu Tacchini, Bento Gonçalves, RS, Brazil