PHYTOCHEMICAL ANALYSIS OF ANTILEISHMANIAL PLANTS: A DRUG
DISCOVERY APPROACH
by
SAMON SHRESTHA
A THESIS
Submitted in partial fulfillment of the requirements for the degree of Master in Science in Chemistry to The School of Graduate Studies of The University of Alabama in Huntsville
HUNTSVILLE, ALABAMA 2015 In presenting this thesis in partial fulfillment of the requirements fora master's degree from The University of Alabama in lluntsville. 1 agree that the Library of this University shall make it freely available for inspection. 1 further agree that permission for extensive copying for scholarly purposes may be granted by my advisor or. in his absence, by the
Chair of the Department or the Dean of the School of Graduate Studies, h is also understood that due recognition shall be given to me and to The University of Alabama in
I Iimtsville in any scholarly use which may be made of any material in this thesis. THESIS APPROVAL FORM
Submitted by Samon Shrestha in partial fulfillment of the requirements for the degreeof
Master ol'Science in Chemistry and accepted on behalf of the Faculty of the School of
Graduate Studies by the thesis committee.
We. the undersigned members of the Graduate Faculty ol'The University of Alabama in
I luntsville. certify that we have advised and/or supervised the candidate on the work described in this thesis. We further certify that we have reviewed thethesis manuscript and approve it in partial fulfillment of the requirements for the degree ofMaster of
Science in Chemistry.
^ThA./, Committee chair
Department Chair
College Dean
Graduate Dean
Ml ABSTRACT
The School of Graduate Studies
The University of Alabama in Huntsville
Dearee Masterof Science College/Dcpt. Science/Chemistry
Name of Candidate Samoa Shrestha
Title 1'hvlochemical Analysis oi'Antileishmanial Plants: A Druti Discovery Approach
Phytochemical analysis of three antileishmanial plants from Monteverde. Costa Ricaand
Abaco Island. Bahamas, has been carried out by use of column chromatography for phytoseparation. and NMR and IR spectroscopy for structural elucidation. Ruyschui phyltadenia contained betulinic acid as major component, with significant amount of lupeol. Eugenia monteverdensis contained (i-Sitosterol. betulinic acid and barbinervic acid. Tahehuia huluunensis contained large amount of ursolic acid. Betulinicacid inhibited promastigotes of A. amazonensis signilleanlly hut was also toxic to mouse maerophages. Lupeoland betulinic acid did not show antimicrobial activity while |5-
Sitosterol inhibited Aspergillus niger strongly, Barbinervic acid expressed good inhibition of Bacillus cereus. Ursolic acid was very active againsi MCF-7 cancer cells and inhibited Staphylococcus aureus significantly. R. phyttadenia and '/'. bahamensis stand out as major sources of medicinally important compounds betulinic acid and ursolic acid respectively.
Abstract Approval: Committee Chair ^>
Department Chair
Graduate Dean
IV ACKNOWLEDGEMENTS
My sincere gratitude goes to my advisor and committee member Dr. William N. Setzer for allowing me to work on this project, his understanding, and guidance with chromatographic techniques and phytochemical analysis. I would like to thank other members of my committee; Dr. Bernhard Vogler for constant helps with NMR use and structural elucidation, and Dr. Robert L. McFeeters for his valuable suggestions and belief in me. The co-operation and support of the committee members were immense and valuable for completion of this project.
I am thankful to the members of Natural Product and Drug Discovery lab for making the time spent in lab enjoyable, especially Bhuwan Chhetri for his help with the chromatographic separation of Tabebuia bahamensis, and Noura Dosoky for performing the cytotoxicity tests. Thanks to Dr. Lianet Monzote and group from Institute of Tropical
Medicine Pedro Kouri for performing the antileishmanial tests.
Support for this work was provided in part by grants from the National Institute of Health
(Grants 1 R15 GM46120-01A1 and 1 R15 CA74343-01). Dr William Haber and Dr.
Robert Lawton helped in plant identification and location. I am thankful to them.
Special thanks to my parents, family and friends for their support and understanding.
v
TABLE OF CONTENTS
Page
List of Figures viii
List of Tables xii
List of Abbreviations xiii
Chapters
ONE INTRODUCTION 1
1.1 Leishmaniasis 1
1.2 Plants and medicines 7
1.3 Plants and leishmaniasis 10
1.4 Plant collection site 16
1.4.1 Monteverde cloud forest 16
1.4.2 Abaco island 19
1.5 Ruyschia phylladenia 20
1.6 Eugenia monteverdensis 23
1.7 Tabebuia bahamensis 28
TWO EXPERIMENTAL 31
2.1 Study area and collection of plants 31
2.2 Antileishmanial screening of plant extracts 34
2.2.1 Antipromastigote assay 34
2.2.2 Antiamastigote assay 34
2.2.3 Cytotoxicity assay (CC50) 35
2.3 Chromatographic separation of Ruyschia phylladenia 36
2.4 Chromatographic separation of Eugenia monteverdensis 38
vi
2.5 Chromatographic separation of Tabebuia bahamensis 39
2.6 IR experiments 41
2.7 NMR experiments 41
2.8 Antimicrobial screening 42
2.9 Cytotoxicity test 43
THREE RESULTS 44
3.1 Compounds from Ruyschia phylladenia 44
3.1.1 Characterization of compound A 44
3.1.2 Characterization of compound B 57
3.2 Compounds from Eugenia monteverdensis 72
3.2.1 Characterization of compound C 72
3.2.2 Characterization of compound D 85
3.2.3 Characterization of compound E 88
3.3 Compounds from Tabebuia bahamensis 100
3.3.1 Characterization of compound F 101
3.4 Result of antileishmanial assay 113
3.5 Results of antimicrobial tests 113
3.5 Results of cytotoxicity assay 113
FOUR DISCUSSION 114
FIVE CONCLUSION 121
REFERENCES 123
vii
LIST OF FIGURES
Figures Page
1.1 Lifecycle of Leishmania spp. 3
1.2 Drugs used in current treatment of Leishmaniasis 6
1.3 Traditionally used medicines from plants 11
1.4 Phytochemicals used traditionally against Leishmaniasis 17
1.5 Cloudzone over Monteverde 21
1.6 Seven lifezones of Monteverde 21
1.7 Abaco Island, Bahamas 22
1.8 Ruyschia phylladenia plant 24
1.9 Leaves and fruits of Ruyschia phylladenia (a) fresh (b) dried 24
1.10 Eugenia monteverdensis tree with fruits 27
1.11 Dried leaves of Eugenia monteverdensis 27
1.12 Leaves of Tabebuia bahamensis 30
1.13 Tabebuia bahamensis plant 30
2.1 Plant collection from Monteverde, Costa Rica 32
2.2 Soxhlet extraction of crude extract 33
2.3 Column chromatography (a) empty cylinder (b) active running column 37
2.4 Chromatographic separation scheme for Ruyschia phylladenia extract 37
2.5 TLC plates under UV light 38
2.6 Chromatographic separation scheme for Eugenia monteverdensis extract 39
2.7 Crystals of SF D after Toluene slow cooling recrystallization 40
2.8 Chromatographic separation scheme for Tabebuia bahamensis extract 41
3.1 Chemical structure of lupeol 44
viii
3.2 Key HMBC correlation of compound A 46
3.3 Proton spectrum of compound A 48
3.4 Proton spectrum of compound A (major peaks) 49
3.5 Carbon spectrum of compound A 50
3.6 Carbon spectrum of compound A (closer view) 51
3.7 HSQC spectrum of compound A 52
3.8 HSQC spectrum of compound A (closer view) 53
3.9 HMBC spectrum of compound A 54
3.10 HMBC spectrum of compound A (closer view) 55
3.11 IR spectrum of compound A 56
3.12 Chemical structure of betulinic acid 57
3.13 Key HMBC and COSY correlations of compound B 59
3.14 Proton spectrum of compound B in chloroform-d 61
3.15 Proton spectrum of compound B in chloroform-d (Major peaks) 62
3.16 Proton spectrum of compound B in DMSO -d6 63
3.17 Carbon spectrum of compound B in chloroform-d 64
3.18 Carbon spectrum of compound B (closer view) 65
3.19 HSQC spectrum of compound B 66
3.20 HSQC spectrum of compound B (closer view) 67
3.21 HMBC spectrum of compound B 68
3.22 HMBC spectrum of compound B (closer view) 69
3.23 COSY spectrum of compound B 70
3.24 IR spectrum of compound B 71
3.25 Chemical structure of β-Sitosterol 72
3.26 Key HMBC and COSY correlations of compound C 74
ix
3.27 Proton spectrum of compound C 76
3.28 Proton spectrum of compound C (Major peaks) 77
3.29 Carbon spectrum of compound C 78
3.30 Carbon spectrum of compound C (closer view) 79
3.31 HSQC spectrum of compound C 80
3.32 HSQC spectrum of compound C (closer view) 81
3.33 HMBC spectrum of compound C 82
3.34 HMBC spectrum of compound C (closer view) 83
3.35 COSY spectrum of compound C 84
3.36 Proton spectrum of compound D 86
3.37 Carbon spectrum of compound D 87
3.38 Chemical structure of barbinervic acid 88
3.39 Key HMBC and COSY correlations of compound E 90
3.40 Proton spectrum of compound E 92
3.41 Proton spectrum of compound E (Major peaks) 93
3.42 Carbon spectrum of compound E 94
3.43 Carbon spectrum of compound E (closer view) 95
3.44 HSQC spectrum of compound E 96
3.45 HSQC spectrum of compound E (closer view) 97
3.46 HMBC spectrum of compound E 98
3.47 HMBC spectrum of compound E (closer view) 99
3.48 COSY spectrum of compound E 100
3.49 Chemical structure of ursolic acid 101
3.50 Key HMBC and COSY correlations of compound F 103
3.51 Proton spectrum of compound F 105
x
3.52 Proton spectrum of compound F (Major peaks) 106
3.53 Carbon spectrum of compound F 107
3.54 Carbon spectrum of compound F (closer view) 108
3.55 HSQC spectrum of compound F 109
3.56 HSQC spectrum of compound F (closer view) 110
3.57 HMBC spectrum of compound F 111
3.58 HMBC spectrum of compound F (closer view) 112
xi
LIST OF TABLES
Tables Page
1.1 Taxonomic classification of pathogenic Leishmania spp. 4
3.1 NMR assignment of compound A (Lupeol) 47
3.2 NMR assignment of compound B (Betulinic acid) 60
3.3 NMR assignment of compound C (β-Sitosterol) 75
3.4 NMR assignment of compound E (Barbinervic acid) 91
3.5 NMR assignment of compound F (Ursolic acid) 104
3.6 Antileishmanial activity (IC50 in μg/ml) of Betulinic acid 113
3.7 Antimicrobial activity (IC50 in μg/ml) of sample compounds 113
3.8 MTT Cytotoxicity Assay result 113
xii
LIST OF ABBREVIATIONS
AIDS…………….Acquired Immune Deficiency Syndrome ATCC……………American Type Culture Collection ATP……………..Adenosine Triphosphate BPH……………..Benign Prostatic Hyperplasia ca………………...circa, approximately CAMHB………….cation-adjusted Mueller Hinton broth
CDCl3……………..deuterated chloroform CFU………………colony forming units cm…………………centimeter(s)
CO2………………..carbon dioxide COSY……………..Correlation Spectroscopy DMSO……………Dimethylsulfoxide DNA…………….Deoxyribonucleic acid EtOAc……………Ethyl acetate GI………………..Gastrointestinal GTP……………..Guanosine-5’-triphosphate HEPES………….4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid HIV……………...Human Immunodeficiency Virus HMBC………….Heteronuclear Multiple Bond Correlation HSQC…………..Heteronuclear Single Quantum Coherence HTS……………..High-throughput screening IC………………..Inhibitory concentration IR……………….Infrared kg……………….kilogram(s) μg……………….microgram(s)
xiii
μl………………..microliter(s) μm………………micrometer(s) mg………………milligram(s) ml……………….milliliter(s) mM……………...millimolar MCF-7…………..Michigan Cancer Foundation-7 MHz…………….megahertz MIC…………….minimum inhibitory concentration MTT…………….3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
NaHCO3………...sodium bicarbonate nm……………….nanometer(s) NMR…………….Nuclear Magnetic Resonance NP(s)…………….Natural Product(s) PBS………………Phosphate buffered saline PKDL……………Post Kala-azar Dermal Leishmaniasis ppm……………...parts per million
Rf…………………Retention factor Spp……………....Species (plural) TLC………………Thin Layer Chromatography UV……………….Ultraviolet w/v……………….weight/volume w/w……………….weight/weight
xiv
CHAPTER ONE
INTRODUCTION
1.1 Leishmaniasis
Leishmaniasis is a group of tropical diseases affecting human and animals, and is characterized by diverse epidemiology, immunopathology and outcome. Various forms of leishmaniasis are caused by about twenty species of Leishmania parasites and transmitted by bite of female sandfly (Phlebotomus spp. and Lutzomyia spp.). This disease remains endemic in several parts of the world, affecting 12 million people in poor, rural, suburban areas of 88 countries in 5 continents, and poses serious public health threats. Most of the people affected are population from third world developing countries (Philippe Desjeux 1996; P. Desjeux 2004; Ouellette, Drummelsmith, and
Papadopoulou 2004). Some general features shared by different forms of this protozoal parasite include: targeting of macrophages, intracellular parasite replication in affected tissue leading to its damage, and the expression and outcome of disease depending upon the host immuno-inflammatory response (Murray et al. 2005).
Leishmania is a genus of flagellated kinetoplastid parasites belonging to
Trypanosomatidae family and is characterized by the presence of kinetoplast which is a
1 distinct part of mitochondria that contains mitochondrial DNA (Chan-Bacab and Peña-
Rodríguez 2001). These parasites have a heteroxenous lifecycle alternating between intracellular amastigotes in the mammalian cells and flagellated promastigotes in the vector (Barrera et al. 2013). The promastigotes which are 15 to 20 μm in length reside in digestive tract of transmitter and infects the vertebrate host by biting. These promastigotes are then phagocytized by the host macrophages where they change to approximately 2.3μm long amastigotes (Berman and Wyler 1980). The transmission of the disease may be anthroponotic where man is the sole reservoir of the parasite, or zoonotic where animal reservoir hosts are also involved (P. Desjeux 2004).
Based on the ability of the protozoa to proliferate in deep tissue or close to the surface of skin, World Health Organization has classified leishmaniasis in four clinical forms:
Cutaneous leishmaniasis: This form of leishmaniasis is caused by L. major and L.
tropica in the Old World (representing Asia, Europe and Africa) and L. mexicana
in New World (representing the Americas). It is characterized by development of
self-healing lesions in skin that evolve from papules to nodules to ulcerative
lesions and may lead to atrophic scars (Herwaldt 1999). The infection by L.
mexicana is relatively benign but that by L. tropica is more severe, chronic and
difficult to treat.
Diffuse cutaneous leishmaniasis: This is a relatively rare type of leishmaniasis
caused by L. amazonensis or L. aethiopica, and is characterized by lesions around
face and external surface of arms, resembling to that of lepromatous leprosy
(Philippe Desjeux 1996; Chan-Bacab and Peña-Rodríguez 2001). It occurs due to
2
defective cell mediated immune response and the lesions do not heal easily and
there is a high chance of relapse.
Mucocutaneous leishmaniasis (Espundia): It is caused mainly by L. braziliensis
and L. panamensis via lymphatic dissemination of amastigote from skin to naso-
oropharyngeal mucus, and is characterized by erosion and disfiguration of mucal
part of nose and mouth.
Figure 1.1 Life cycle of Leishmania spp. (“Morphology and Life Cycle” 2015).
3
Visceral leishmaniasis (kala-azar): It is the most severe and fatal form of the
disease, caused by L. donovani, and is characterized by its effect in internal
organs like liver, spleen and bone marrow. The incubation period varies from
weeks to months and common symptoms include irregular fever, weight loss and
anemia. A major risk associated with this form of leishmaniasis is post-recovery
development of Post Kala-azar Dermal Leishmaniasis (PKDL) by dissemination
of residual reservoir of the parasite.
Cutaneous leishmaniasis affects about 1-1.5 million people and visceral leishmaniasis affects about 0.5 million children and adults, causing about 70,000 deaths
Table1.1 Taxonomic classification of pathogenic Leishmania spp. (Chan-Bacab and Peña-Rodríguez 2001)
4 every year (Murray et al. 2005). Of total, 90% of cutaneous leishmaniasis infections occur in Afghanistan, Pakistan, Syria, Saudi Arabia, Algeria, Iran, Brazil and Peru while the same percentage of visceral leishmaniasis occur in India, Bangladesh, Nepal, Sudan and Brazil (Philippe Desjeux 1996; Murray et al. 2005). Most of the population in these countries is fighting poverty which amplifies the risks and problems of the disease. In
USA, leishmaniasis is an emerging zoonosis, especially among US soldiers in the
Middle-East and peace-keeping corps, with about 500 confirmed cases (Ouellette,
Drummelsmith, and Papadopoulou 2004).
The current treatments of leishmaniasis include the use of toxic heavy metals like antimony. Pentavalent antimonial like sodium stibugluconate and meglumine antimonate constitute the first line drug against the disease since 50 years (Salem and Werbovetz
2006). These antimonial work by reducing the net generation of ATP and GTP by binding to various enzymes associated with glycolysis and oxidation of fatty acids, and inhibits energy production in amastigotes (Berman 1988). Another recent study suggests that antimony inhibits the thiol redox potential of the parasite cell by efflux of intracellular thiol and by hindering trypanothione reductase (Ouellette, Drummelsmith, and Papadopoulou 2004). Second line treatment includes use of Amphotericin B,
Pentamidine, and recently, oral Miltefosine has been approved for human visceral leishmaniasis. Pentamidine acts by binding to DNA of parasite and inhibiting them and
Amphotericin B binds to ergosterol fraction of parasites’ cell membrane, increasing its permeability and leading to its death (Ouellette, Drummelsmith, and Papadopoulou 2004;
Chan-Bacab and Peña-Rodríguez 2001). Miltefosine has been known to inhibit
P13K/Akt signaling pathway in the parasite (Ogungbe and Setzer 2013).
5
Sodium stibugluconate Meglumine antimonate
Amphotericin B
Pentamidine
Fig 1.2 Drugs used in current treatment of Leishmaniasis.
6
These current treatments, however, have lots of side effects and drawbacks associated with them that limit their uses.
Antimonial and Pentamidine are associated with side effects like stiff joints,
cardiotoxicity, gastrointestinal (GI) distress, hepatic and renal insufficiency.
Amphotericin B leads to side effects that include alteration of renal function
(Herwaldt 1999).
Parenteral administration of drugs and requirement for long duration of
administration are major limitations.
The diagnosis and treatment regime are too expensive for people of low income in
developing countries. For example, in Nepal where majority of people in rural
areas are living in poverty, the cost per death averted for leishmaniasis sums up to
US$ 131 in outreach program and US$ 200 at health facility (P. Desjeux 2004).
Increasing resistance against antimonials has been reported in northeast India
(Croft and Yardley 2002).
These limitations of chemotherapy and failure of vaccination approach to enter clinical trial (Polonio and Efferth 2008; Mishra et al. 2009) and inability of total vector control calls for a more efficient and accessible mode of treatment against leishmaniasis.
1.2 Plants and medicines
Natural Products (NPs) represent a huge, mostly untapped source for modern medicines. The structural complexity and clinical specificity of biologically active molecules, produced as a result of evolutionary pressure, maintains NPs as the prototype for major chemicals used as drugs (Harvey 2007). NPs have been used as medicines for
7 thousands of years, as their formation indicates selectable evolutionary advantages to the producing organism. Various traditional medicines obtained from plants were the basis of early medicines like morphine, aspirin, digitoxin, quinine and pilocarpine (Lam 2007;
Butler 2004). Besides plants, other NP sources of medicines include microbes, which is the major source of antibiotics, animals, and marine environment which has generated interest as the largest potential source of biodiversity and novel chemicals with useful bioactivity (Harvey 2007; Harvey 2008).
Around 80% of medicinal products used today were either directly derived or inspired from NPs (Harvey 2007). Between 2001 and 2005, 23 new NPs based drugs were introduced for treatment of a range of therapeutic conditions including cancer, infections, Alzheimer’s disease and diabetes among others (Lam 2007). Between 2005 and 2010, 7 NPs, 10 semi-synthetic NPs and 2 NPs derived drugs were approved for marketing worldwide (Mishra and Tiwari 2011). However, in the late 20th and early 21st centuries, big pharmaceutical companies had abandoned their interest in NP drug discovery and turned their attention to high-throughput screening (HTS) and combinatorial synthesis with the idea of preparing large libraries of synthetic compounds for quick development of new drugs. Due to drawbacks of NP drug discovery that included decreasing success rates, possibility of very small, unusable amount of bioactive substance, difficulty in extraction, separation and identification of active compounds, and competition among pharmaceutical industries to come up with new drugs first, pharmaceutical industries opted for quicker alternative routes (J. W. H. Li and Vederas
2011; Harvey 2008). The idea was that combinatorial chemistry would generate libraries containing millions of compounds, which would be screened by HTS to produce drug
8 leads (Butler 2004). Although initially this idea seemed a better one, the failure of these alternative routes to deliver many lead compounds in major therapeutic issues like immuno-suppression, infective diseases and metabolic diseases saw the renewed interest in natural product researches. The number of new, bioactive synthetic chemical entities reached its minimum in 20 years in 2002 (Ortholand and Ganesan 2004). Since then, the combination of NPs and synthetic chemistry is considered a better route and with development of technologies and new methods, the screening of NPs has eased and they have been re-established as the major source of drug leads (M. C. Setzer et al. 2003).
Shan Hai Ching, one of the earliest surviving pharmaceutical texts dating back to
250 B.C., describes the first medicinal use of plants (Chen, Lee, and Kuo 1993; Ding and
Ceng 1985). The production of morphine from poppy (Papaver somniferum) by
Friedrich Serturner in 1804 A.D. initiated the era of diverse study, purification, and administration of drugs obtained from plants (J. W. H. Li and Vederas 2011). The current medical world has seen the use of plants in treatment and cure of almost all the diseases. Some common examples include antimalarial drug artemisinin obtained from
Artemisia annua (Shu 1998); cholinesterase inhibitor physostigmine obtained from
Physostigma venenosum; Huperzine A used against Alzheimer’s disease, obtained from
Huperzia serrata (Mishra and Tiwari 2011); anticancer drugs like paclitaxel (Taxol®) from Taxus brevifolia (Rowinsky, Cazenave, and Donehower 1990), etoposide from
Podophyllum peltatum (Damayanthi and Lown 1998), vinblastine from Catharantus roseus (Owellen et al. 1976); anti-hypertensive drugs digoxin and digitoxin obtained from Digitalis purpurea, and anticholinergic atropine obtained from Atropa belladonna.
Calanolide obtained from Malaysian rainforest tree Calophyllum lanigerum is currently
9 under clinical development as a potential treatment against AIDS and HIV infection (Shu
1998).
1.3 Plants and leishmaniasis
Due to the neglect of leishmaniasis by big pharmaceutical industries, and due to limited availability of few effective medicines developed against this disease, most patients in leishmaniasis endemic areas depend on traditionally used native plants to alleviate the symptoms. Folk medicine is very good source of effective treatment for this disease as well, like numerous others mentioned above. In 1984, based on the ethno- medicinal knowledge of the local population, a plant screening program for potential leishmanicides was started in French Guiana (Rocha et al. 2005). The rainforests of
South America and Africa has been the source of tens of potential antileishmanial products in the past 5 years (De Carvalho and Ferreira 2001). There are numerous examples in the literature that have mentioned the use of native plants, in the form of crude extract, orally or as topical preparation for skin infections of leishmaniasis. Several groups of researchers have published numerous reviews of plant used traditionally against leishmaniasis (Polonio and Efferth 2008; Akendengue et al. 1999; De Carvalho and Ferreira 2001).
Alkaloids are nitrogen-containing heterocyclic compounds widely distributed among many groups of plants. Many alkaloids have been found to show antileishmanial activity. Several aryl and alkyl-2 quinoline alkaloids obtained from Galipea longiflora have been found to be active against different species of Leishmania. These include chimanine B, chimanine D, 2-n-propyl quinoline active against L. braziliensis
10
i ii
iii
iv v
vi
Fig 1.3 Traditionally used medicines from plants i) Morphine ii) Aspirin iii) Digoxin iv) Quinine v) Pilocarpine vi) Artemisinin vii) Physostigmine viii) Huperzine A ix) Taxol x) Etoposide xi) Vinblastine xii) Atropine
11
vii viii
ix x
xi xii
Fig 1.3 (continued)
12 promastigotes (Akendengue et al. 1999; Polonio and Efferth 2008; De Carvalho and
Ferreira 2001). Berberine, a common quaternary isoquinoline alkaloid, found in
Berberidaceae and Annonaceae family is one of the alkaloids with highest antileishmanial activity, and is known to inhibit L. major amastigotes, and its derivatives tetrahydroberberine is more potent against L. donovani (Sen and Chatterjee 2011; Salem and Werbovetz 2006; Akendengue et al. 1999). Other leishmanicidal alkaloids include coronaridine and voacangine from Tababaemonta catharinensis (Sen and Chatterjee
2011), and cephaeline obtained from Psychotria klugii. These are active against L. donovani (Salem and Werbovetz 2006).
Flavonoids, obtained from flavones, are water soluble plant compounds with two benzene rings separated by a propane unit, and are generally found in plants as their glycosides. These are phenolic compounds and thus possess hydroxyl groups (-OH group) attached to the aromatic ring. Flavonoids like luteolin and quercetin, isolated from Vitex negundo, are known to inactivate L. donovani promastigotes by initiating morphological changes and causing loss of cellular integrity leading to cell cycle arrest in
G1 phase (Polonio and Efferth 2008; Sen and Chatterjee 2011). Other flavonoids with antileishmanial activity include isoorientin, flavone A and isoflavans like 8- prenylmucronulatol, glyasperin H, and smiranicin from Smirnowia iranica (Salem and
Werbovetz 2006).
Chalcones represent a class of flavonoids that lack pyran rings found in flavonoids, and are more fully conjugated. Licochalcone A, an oxygenated chalcone, found in the roots of Chinese liquorice has shown to inhibit L. major amastigotes by damaging the mitochondria of the parasites. Other chalcones showing antileishmanial
13
,methoxychalcone obtained from Piper aduncum-׳Dihydroxy-4-׳6,׳activities include 2
dimethyl-chalcone from Psorothamnus polydenius-׳5,׳methoxy-3-׳trihydroxy-6-׳4,׳2,2
(Polonio and Efferth 2008; Salem and Werbovetz 2006).
Quinone is another class of phenolic compound that play important role in respiration of plants. Quinones like plumbagin from Plumbago sp. and diospyrin from
Diospyros montana show potent activity against L. donovani promastigotes (Polonio and
Efferth 2008; Salem and Werbovetz 2006).
Terpenes are hydrocarbon–based natural products that possess considerable amount of structural diversity, but are all derived from common isoprene units. Several terpenes have also been found to show antileishmanial activity. Linalool, a monoterpenic
(10 carbon containing terpene) alcohol constituent of many essential oils, has shown potent antileishmanial activity against L. amazonensis promastigotes and amastigotes
(Sen and Chatterjee 2011; Salem and Werbovetz 2006). Espintanol, a monoterpenoid obtained from bark of Oxandra espintana has been known to show significant activity against promastigotes of twelve species of Leishmania, including L. mexicana (Chan-
Bacab and Peña-Rodríguez 2001). Parthenolide, a sesquiterpene (15 carbon containing terpene) lactone obtained from Tanacetum parthenium has shown significant activity against L. amazonensis promastigotes and amastigotes. Anti-malarial drug artemisinin isolated from Artemisia annua was recently tested to show potent activity against the parasites’ amastigotes (Polonio and Efferth 2008). Jatrogrossidione obtained from
Jatropha grossidentata and jatrophone from Jatropha isabellii are diterpenes (20 carbon containing terpenes) active against promastigotes of L. braziliensis, L. amazonensis, and
L. chagasi (Chan-Bacab and Peña-Rodríguez 2001). Simalikalactone D isolated from
14 root bark of Simaba orinocensis is a decanortriterpenoid that has shown potent activity against L. donovani promastigotes, and common triterpenes (30 carbon containing triterpenes) like ursolic acid and oleanolic acid have shown antileishmanial activity against L. amazonensis (Salem and Werbovetz 2006). Dihydrobetulinic acid, an abundantly available triterpene causes apoptosis in L. donovani (Sen and Chatterjee
2011). Saponins are high- molecular weight triterpene glycosides with the sugar part
(glycone) attached to non-sugar triterpene or sterol (aglycone). Saponins like α-hederin,
β-hederin and hederacolchiside A1 obtained from Hedera helix showed strong antileishmanial activity against all life stages of L. infantum and L. mexicana by altering membrane intensity and membrane potential. A steroidal saponin racemoside A, isolated from Asparagus racemosus shows antileishmanial activity against L. donovani promastigotes and amastigotes (Polonio and Efferth 2008).
Several ellagitannins and proanthocyanidins have shown antileishmanial activity by inducing potent tumor necrosis factor that inhibited the intracellular amastigotes but did not affect the promastigotes or mammalian host cells (Salem and Werbovetz 2006).
Other groups of compounds with antileishmanial activity include iridoids like amarogentin from Swertia chirata, and lignans like diphyllin isolated from Haplophyllum bucharicum.
Most of these antileishmanial products act by targeting several proteins in
Leishmania that are essential for metabolism of glucose (e.g. pyruvate kinase, phosphoglucose isomerase, glyceraldehydes-3-phosphate dehydrogenase), sterols, nucleotide ( dihydroorotate dehydrogenase, deoxyuridine triphosphate nucleotidohydrolase, nicotinamidase), glycosylphosphatidylinositol and enzymes
15 essential for maintenance of trypanothione and polyamine levels (Ogungbe and Setzer
2013).
Despite these many numbers of plant products showing antileishmanial activity, the search for other antileishmanial products from plants is still ongoing and necessary.
The main reason for this is that many of these natural products do not meet all the criteria essential for their development into a commercial drug. The new drug must be cheap for the affected group of people to afford it. This requires the new antileishmanial natural product to be easily and sufficiently available so that the cost product for drug manufacturing is less. Other requirements include the absence of severe side effects, ease of use, and selectivity between human macrophages and parasites. Most of all, it is essential to find the natural products which along with having the above mentioned requirements, must be active against the amastigote phase of the parasite as this is the form that affects humans. Hence, the quest for antileishmanial drug from natural products is still an ongoing process.
1.4 Plant collection site
Ruyschia phylladenia and Eugenia monteverdensis were collected from
Monteverde, Costa Rica, and Tabebuia bahamensis from Abaco Island, Bahamas.
1.4.1 Monteverde cloud forest
Monteverde region, located in Northwestern Costa Rica, on the upper pacific slope of mountain range Cordillera de Tilaran, is geographically and climatically diverse like most tropical montane areas (Clark, Lawton, and Butler 2000). Based on average temperature and rainfall, Holdridge has described Monteverde as having 7 of total 12 life
16
Berberine Chimanine B Chimanine D
17
Luteolin Quercetin Flavone A
methoxychalcone-׳dihydroxy-4-׳6 ,׳Smiranicin Licochalcone A 2
Fig: 1.4 Phytochemicals used traditionally against Leishmaniasis
Plumbagin Diospyrin Isoprene unit Linalool
18
Parthenolide Jatrogrossidione Ursolic acid Oleanolic acid
Dihydrobetulinic acid
Fig: 1.4 (continued)
zones of Costa Rica. The seven life zones are tropical moist forest (700-800 m on Pacific slope), premontane moist forest (700-1200 m on Pacific slope), premontane wet forest
(800-1500 m on Pacific slope), lower montane wet forest (1450-1600 m on Pacific slope), lower montane rain forest (1550-1850 m on Continental Divide), premontane rain forest (700-1400 m in Atlantic slope), and tropical wet forest (700 m on Atlantic Side)
(Fig. 1.7). These zones exhibit wide variation of vegetation with tropical moist forest zone full of mostly evergreen plants; premontane wet forest, where it rains almost every day, possessing relatively few species; constantly misty lower montane region with wet forest perfect for epiphytes; lower montane rainforest with very dense vegetation and having heavy and constant mist and cloud cover; premontane rainforest rich with dense evergreen forest, high humidity and abundant rainfall (Nadkarni 2000). These environmental variations in the Monteverde region has set up Monteverde cloud forest as a rich source of biodiversity and one of the floristically most diverse places in the world.
The slopes of Cordillera above 1200 m elevation contain approximately 1700 plant species and the area above 700 m in Cordillera de Tilaran contains approximately
3000 plant species (M. C. Setzer et al. 2003). Most of these plants have not been explored for their phytochemical properties as the region above 1500 m was never settled because of unfavorable environmental conditions, difficulty for agriculture and remoteness.
1.4.2 Abaco island
Abaco Island is a part of Commonwealth of Bahamas, and is located in the Little
Bahama Bank, in the most northeastern part of the archipelago which consist 29 islands in total. The Abaco Island whose landmass consists of two main islands, Great Abaco
19
Island and Little Abaco Island, is bordered on the east by deep waters of Atlantic Ocean, on the south by the deep waters of N.W. Providence Channel and N.E Providence
Channel and on the west by shallow waters of Little Bahamas Bank (Walker et al. 2008).
The temperature of the island remains well above freezing in winter and is tropical in the summer. The northern islands receive high precipitation, especially in summer, with an average 1400 cm of rainfall every year (Dodge 1995). The island holds a wide variety of vegetation because of its high precipitation.
The majority of these vegetation from both collection sites hold a large potential for untapped resources that are rich in pharmacologically important chemicals (M. C.
Setzer et al. 2003; Farnsworth 1988; Eisner 1990). These plants have survived millions of years of competition from numerous parasites, pathogens, microbes (like bacteria, fungi), insects, nematodes, and even herbivores. This indicates the presence of plethora of diverse defensive chemicals and their derivatives in these plants. These chemicals are strong candidates for medicines that can be extracted and many of them can be successfully used for treatment of several human diseases.
1.5 Ruyschia phylladenia
Ruyschia phylladenia Sandwith is a neotropical plant belonging to
Marcgraviaceae family that is found commonly in Costa Rica and Panama at an altitude of about 1200m (Lens et al. 2005; Machado and Lopes 2000). Seven different species of
Ruyschia have been accepted till now (Roon 2005) . R. phylladenia Sandwith is a vine on pasture tree with cream-green petals and brown anthers. The fruits are about 3 mm in size and green tinged with red in color (“Flora Mesoamericana” 2015).
20
Fig 1.5 Cloudzone over Monteverde
Atlantic
Pacific
Fig 1.6 Seven lifezones of Monteverde (“Maps of Monteverde, Costa Rica” 2015)
21
Fig 1.7: Abaco Island, Bahamas
The Marcgraviaceae is a family of 130 species of plants distributed over seven different genera; Marcgravia, Souroubea, Ruyschia, Marcgraviastrum, Sarcopera,
Norantea, and Schwartzia. This family includes mainly lianas, climbing shrubs and treelets ranging from northern Bolivia and southern Brazil to southern Mexico (Gentry
1994). This family is characterized by several sclereids, hypophyllus glands and nectar bracts which attract birds, insects and mammals like bats as a source of pollination
(Kubitzki 2004). The genera Souroubea holds medicinal importance traditionally. South
American species of Souroubea are mentioned in “The Healing Forest” with S. guianensis Aubet var Coralline’s dry leaves boiled to prepare calming drinks, and S. guianensis Aubet var cylindrica used to treat ‘susto’ or fright as tranquilizer (Puniani et al. 2014; Schultes and Raffauf 1990). The bark of the S. sympetala was used as decoction to treat fever, ulcer and dysentery and against witchcraft by traditional healer of Indian creek, Belize. The plant extract of S. sympetala (Mullally et al. 2011) and S.
22
gilgii showed excellent anxiolytic activity in standardized elevated test maze, supporting the ethnobotany (Puniani et al. 2014). The dried leaves of S. pachyphylla Gilg. are applied as ointment to the eyes in case of infection and conjunctivitis in several parts of
Columbia (Quattrocchi 2012) .
The genera Ruyschia and Souroubea are closely related and were considered to be the same in the past, with several species of Ruyschia placed under Souroubea.
However, the two species were later separated and considered independent based on the difference of their nectariferous bract and pistil. Ruyschia has small, foliaceous, flat or gibbous to slightly concave nectariferous bracts while Souroubea has hollow, cup or spur shaped and auriculate nectariferous bracts. The former has a subglobular, mostly–loculed ovary with a minute and distinct style and a small stigma while the latter possess an often angular, 3 to 5 loculed ovary with a large, sessile, often radiate stigma (Roon 2005).
Considering the similarity, it can be speculated that Ruyschia phylladenia has some medicinally important compounds as well. There is no literature published in the phytochemical analysis of Ruyschia phylladenia till the time of this writing.
The acetone bark extract of this plant showed antileishmanial activity of <12.5
μg/mL against promastigotes and 22.0 ± 5.9 μg/mL against amastigotes of L. amazonensis while being non-toxic to mouse macrophages (Monzote, Piñón, and Setzer
2014).
1.6 Eugenia monteverdensis
Eugenia monteverdensis is a recently described species of Eugenia, belonging to
Myrtaceae family. The Myrtaceae consists of around 129 genera and 4620 species of plants, of which Eugenia is one of the largest genera with about 500 species
23
Fig 1.8 Ruyschia phylladenia plant
(a) (b)
Fig 1.9 Leaves and fruits of Ruyschia phylladenia (a) fresh (b) dried
24
(Cole, Haber, and Setzer 2007; Mabberley 1997). E. monteverdensis is a tree that grows up to 20 m and have characteristic leaves that is elliptic or obovate, with margins strongly revolute near the petiole, and fruits that are yellow, irregularly obovoid or ellipsoid and lacks ribbing (Barrie 2005). The tree is local to montane forests of central Costa Rica at elevation of 1000-2000 m. Several species of Eugenia have been reported for use as traditional medicines. E. uniflora is a shrubby tree that has been found to be medicinally important in conditions like fever, inflammation, stomach diseases, hypotension, gout and hypoglycemia (Kanazawa, Patin, and Greene 2000; Ogunwande et al. 2005; Cole, Haber, and Setzer 2007; Lago et al. 2011). E. sandwicensis has been reported to be potent cytotoxic agent (Gu et al. 2001). Another medicinally important Eugenia species include
E. caryophyllata which is traditionally used in different parts of Asia and Africa to treat toothache, earache, arthritis pain, cold, gum diseases and also as anti-fungal, anti- rheumatic, anticonvulsant, and anticarcinogenic (Nyegue et al. 2014; Joshi et al. 2011).
The seed of E. jambolana, commonly known as Jamun in Nepal and India, is used as hypoglycemic, anti-inflammatory, anti-bacterial, anti-diarrheal, anti-ulcerative, antioxidant and hypolipidemic (Ravi, Ramachandran, and Subramanian 2004; Sarita and
Bhagya 2012; Gao et al. 2009). These activities are attributed to its rich constituents of flavonoids, tannins, gallic acids, ellagic acids and glycosides. E. brasiliensis Lamarck is used traditionally as anti-rheumatic, anti-diarrheal, and as diuretic in Brazil (Lima et al.
2008; M. DA Magina et al. 2009). E. beaurepaireana (Kiaerskou) is used in South
America as an astringent, anti-inflammatory and anti-ulcerative. E.umbelliflora (Berg.) leaf extract shows strong antibacterial effect ( Magina et al. 2009). Some other Eugenia spp. used as traditional medicines include E. axillaris (aphrodisiac, diarrhea), E.
25
cauliflora (asthma, dysentery), E. citrifolia (migraine, cardiac problems), E. dysenterica
(dysentery, kidney and bladder diseases), and E. punicifolia (flu, antidiabetic, liver disease, wounds, infections) (Stefanello, Pascoal, and Salvador 2011).
Ethanol bark extracts of E. austin-smithii and Eugenia sp. from Alberto Manuel
Brenes Biological Reserve, Costa Rica, has shown antileishmanial activity. Hexane and methanol fruit extracts of E. umbelliflora were leishmanicidal to L. amazonensis and L. braziliensis promastigotes. Ethanol leaf extract of E. uniflora was marginally active against L. braziliensis (Monzote, Piñón, and Setzer 2014).
Besides Eugenia spp., various other plants belonging to Myrtaceae family are used as folk medicines too. The common ones include Psidium guajava whose leaves hold importance against conditions like inflammation, pulmonary disease, cough, vomiting and diarrhea. Acca sellowiana is a bushy shrub found in South America whose fruit show bactericidal activity against Gram-negative bacteria (Lapčík et al. 2005;
Vuotto et al. 2000). Syzygium guineense is a small tree whose bark is traditionally used to treat stomachache and diarrhea in Subsaharan Africa (Djoukeng et al. 2005). Leaves of Eucalyptus tereticornis has been used as mosquito repellent for long (Senthil Nathan
2007).
Cole et al. studied the leaf essential oil of E. monteverdensis and found it to be rich in oxygenated monoterpenoids, fatty acid derivatives and oxygenated sesquiterpenoids, with a smaller quantity of sesquiterpene hydrocarbons. The plant essential oil is rich in compounds like linalool, trans-2-hexenal, trans-pinocarveol, and α- terpineol (Cole, Haber, and Setzer 2007). The acetone bark extract of this plant showed
26
Fig 1.10 Eugenia monteverdensis tree with fruits
Fig 1.11 Dried leaves of Eugenia monteverdensis
27
antileishmanial activity of 23.9 ± 2.8μg/mL against promastigotes and 20.7 ± 4.5 μg/mL against amastigotes of L. amazonensis while being non-toxic to mouse macrophages
(Monzote, Piñón, and Setzer 2014).
However, there has not been any study in the phytochemical analysis of this plant’s extract, and any published study on the medicinal importance of this plant is not available. Hence the phytochemistry of Eugenia monteverdensis is unknown.
1.7 Tabebuia bahamensis
Tabebuia bahamensis, commonly known as “Five Fingers” is a small tree belonging to Bignoniaceae family that is common in Bahamas. The Bignoniaceae family consists of 110 genera and 650 species of flowering plants, of which about 100 belong to
Tabebuia genera, and is predominantly found in tropical and subtropical countries
(Rahmatullah et al. 2010; Ospina, Buendia, and Ibarra 2013). T. bahamensis possess small leaves with leaflets that are slender with green color on the upper surface and strongly whitish lower surface. It is a slim tree with straight upright branches. It grows well in properly drained, alkaline or acidic soils. The leaves possess 3-5 leaflets and flowers vary in color from white to pink. It blossoms throughout the year (Popenoe
1980).
Several species of Tabebuia holds ethnopharmacological and ethnobotanical importance. The leaf decoction of T. bahamensis was used as sex stimulant and aphrodisiac and the leaf decoction of T. heterophylla was used as anti-gonorrheal in the
Caribbean (Halberstein 2005). T. bahamensis was an essential component of ‘love potion’ prepared in Andros Island of Bahamas (Eshbaugh 2014). T. rosea (Bertol.) is widely used as Thai traditional medicine as the tree extract shows antimicrobial, anti-
28
inflammatory, anti-bacterial, antifungal, diuretic and laxative properties (Sichaem et al.
2012). The bark of T. avellanedae, a plant that has been known for its medicinal importance since Incan era, is used externally as poultice or concentrated tea for treatment of skin inflammatory diseases like eczema, psoriasis and fungal infection in northeast region of Brazil (Suo et al. 2012; Byeon et al. 2008). This plant has been used as an astringent, anticoagulant, against anxiety, poor memory, irritability, and even for the treatment of cancer in folk medicine (Yamashita et al. 2007; Freitas et al. 2013).
Another plant of Tabebuia genera with great ethnopharmacological importance is T. impetiginosa. The stem bark of this plant has been used in North and South America as anticancer, antifungal, anti-inflammatory and antibacterial (Koyama et al. 2000). The bark of this plant has also been used for treatment of diabetes, fever, malaria, stomach disorders, ulcers and syphilis (Warashina, Nagatani, and Noro 2004; Gómez Castellanos,
Prieto, and Heinrich 2009). T. aurea, known as Paratudo, is used traditionally as infusion or maceration with alcohol for treatment of snake bites and also as anti-inflammatory
(Reis et al. 2014). T. argentea, common to South America, is used as a folk medicine against inflammation, influenza, whooping cough, asthma and spasmodic cough (De
Abreu et al. 2014).
Monzote et al. studied the acetone bark extract of T. bahamensis (Northr.) Britton against L. amazonensis macrophages and determined its IC50 to be 17.4±3.5 μg/mL for promastigotes and 23.8±5.7 μg/mL for amastigotes. The extract showed CC50 of >200
μg/mL against mouse macrophage proving to be good candidate for drug against L. amazonensis (Monzote, Piñón, and Setzer 2014).
29
Fig 1.12 Leaves of Tabebuia bahamensis
Fig 1.13 Tabebuia bahamensis plant
30
CHAPTER TWO
EXPERIMENTAL
2.1 Study area and collection of plants
Ruyschia phylladenia and Eugenia monteverdensis were collected from
Monteverde Cloud Forest Reserve (ca. 1600 m elevation), the community of Monteverde
(ca.1400 m elevation), or from the San Luis Biological Station (ca. 700 m elevation) and
Tabebuia bahamensis was collected from Abaco Island, Bahamas by William N. Setzer and group. The plants identification was confirmed by Robert O. Lawton or William A.
Haber by sample comparison at Missouri Botanical Garden. All voucher specimens are currently at Missouri Botanical Garden. The plant materials were collected, chopped and immediately extracted, initially by infusion of approximately 100 g of finely chopped material in either a mixture of ethanol/chloroform (1:1) or in acetone at ambient temperature for 48 hours. The samples that showed activity in this screening were collected in quantity of 1-2 kg and chopped and extracted by Soxhlet extraction with refluxing acetone or DCM for 4 hours by William N. Setzer and group. The extract, about 25 grams, were then stored in an amber colored jar in a freezer at 4̊C until use.
31
(a)
(b)
Fig 2.1 Plant collection from Monteverde, Costa Rica
32
Fig 2.2 Soxhlet extraction of crude extract
33
2.2 Antileishmanial screening of plant extracts
The antileishmanial screening was performed by Lianet Monzote and Abel Piñón at Parasitology Department, Institute of Tropical Medicine “Pedro Kourï”.
2.2.1 Antipromastigote assay
50 μL of Schneider’s medium was put in each well of 96 well plate. 48 μL of medium and 2 μL of test extracts were added in the first well of each lane. Extract solution were diluted serially (1:1) along each lane of the plate with medium by removing
50 μL of test solution and adding 50 μL of medium. Then, 50 μL of exponentially growing L. amazonensis (MHOM/77BR/LTB0016) promastigote cells, at a concentration
5 of 2 x 10 promastigotes/mL were added to each well to give a final concentration ranging from 12.5 to 200 μL/mL. After an incubation of 72 hours at 26 ̊ C, 20 μL of freshly prepared (5mg/mL in Phosphate Buffered Saline, PBS), filtered and sterilized 3-
[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) (SIGMA, St. Louis,
MO, USA) was added to the wells. After 4 hours of further incubation, 100 μL of
Dimethylsulfoxide (DMSO) were added to dissolve the formazan crystals. The optical density was determined using a spectrophotometer at a test wavelength of 560 nm and a reference wavelength of 630 nm and medial inhibition concentrations were calculated.
2.2.2 Antiamastigote assay
The mouse peritoneal macrophages were harvested and plated at concentration of
106 cells/mL in 24-well Lab-Tek plates (Costar®, Washington, DC, USA), and incubated at 37 °C under an atmosphere of 5% CO2 for 2 hours. PBS was used to wash away the non-adherent cells. Stationary-phase of L. amazonensis promastigotes were added at a
34
4:1 parasite/macrophage ratio to each well and the cultures were incubated for 4 hours more. The free parasites were then removed by washing the cell monolayers with PBS three times. Then, to each well, 1990 μL of RPMI (Roswell Park Memorial Institution) completed medium and 10 μL of plant extracts dissolved in DMSO were added. Four dilutions of 1:2 ratios were carried out by taking 1000 μL each time to obtain test concentration of extracts ranging from 12.5 to 100μg/mL, in duplicate cultures, followed by incubation for further 48 hours. The parasites were then fixed in absolute methanol, stained with Giemsa, and examined under light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 25 macrophages per sample, and the results were expressed as percent of reduction of the infection rate in comparison to that of the controls. The infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages and the
IC50 value was determined.
2.2.3 Cytotoxicity assay (CC50)
The toxicity of the extracts was tested against mouse peritoneal macrophages which act as the host for the amastigotes form of the parasites. The macrophages were collected from peritoneal cavities of normal BALB/c mice in ice-cold RPMI 1640 medium, supplemented with antibiotics, seeded at 30,000 cells/well, and incubated for 2 hours at 37̊ C in 5% CO2 environment. PBS was used to get rid of non-adherent cells, and to each cell, 2 μL of extracts in 98 μL of medium with 10% FBS (Fetal Bovine
Serum) and antibiotics were added. The test concentrations ranging from12.5 μg/mL to
200 μg/mL were obtained by serial dilution and the cells were incubated with test solutions for 72 hours. Colorimetric assay with MTT was used to determine the
35
cytotoxicity by adding 15 μL of MTT to each well, incubating for 4 hours and checking the optical density after dissolving the formazan crystals with 100 μL DMSO.
2.3 Chromatographic separation of Ruyschia phylladenia
About 25 grams of acetone bark extract of Ruyschia phylladenia was subjected to bioactivity-directed column chromatography using silica (40-63 μm particle size and 60
Å porosity) as the stationary phase in a column of length 90 cm (length) × 5 cm
(diameter). The elution was carried out using hexane/ethyl acetate (EtOAc) step gradient as illustrated in figure 2.4. Each solvent system was used at a volume of 500 mL to 1L based on the movement of the extract through column by the solvent.
The different fractions were analyzed with thin layer chromatography (TLC) by spotting few drops of concentrated fraction samples and viewing the TLC plates (200 μm thickness) under UV light after developing the plates. The fractions with similar retention factor (Rf) value were combined to obtain superfractions SF(s). Fraction 13 and
14 formed SF A and fraction 24 through 42 formed another SF B.
The SFs were then purified by recrystallization. Both the SFs were recrystallized by vapor diffusion method using EtOAc and pentane solvents. For this, a small amount of the fraction in a vial was dissolved in small volume of EtOAc by gentle heating and the vial was placed in a larger vial with pentane. The set up was kept undisturbed for a few days to obtain crystals of respective fractions. The crystals were then separated from the supernatant. The purified crystals were dissolved in deuterated solvent (7.5 mg in 750
μL) and NMR experiments were run.
36
(a) (b)
Fig 2.3 Column chromatography (a) empty cylinder (b) active running column
.
Fig 2.4 Chromatographic separation scheme for Ruyschia phylladenia extract
37
Fig 2.5 TLC plate under UV light
2.4 Chromatographic separation of Eugenia monteverdensis
About 25 grams of acetone bark extract of Eugenia monteverdensis was subjected to bioactivity-directed flash chromatography using silica (40-63 μm particle size and 60
Å porosity) as the stationary phase in a column of length 90 cm (length) × 5 cm
(diameter). The elution was carried out using hexane/EtOAc step gradient as illustrated in figure 2.6. Each solvent system was used at a volume of 500 mL to 1L based on the movement of the extract in column by the solvent.
The different fractions were analyzed by thin layer chromatography (TLC) by spotting few drops of concentrated fraction samples, letting the plate develop, and viewing the TLC plates under UV light. The fractions with similar retention factor (Rf) value were combined to obtain SFs. Fractions 31 through 36 formed SF C, fraction 72 and 73 formed a SF D and fraction 105-106 formed SF E.
38
Fig 2.6 Chromatographic separation scheme for Eugenia monteverdensis extract
The SFs were then purified by recrystallization methods. The SF C was recrystallized by toluene slow cooling method. The sample was dissolved in a small amount of toluene by heating to form a saturated solution. The setting was placed undisturbed at 4̊ C for a few days to let the crystals develop. Finally, the crystals were separated from the supernatant. The SF D and E were recrystallized by vapor diffusion method using EtOAc/ pentane. All the samples were dissolved in suitable deuterated solvent and NMR experiments were run to determine the structures.
2.5 Chromatographic separation of Tabebuia bahamensis
About 25 grams of dichloromethane leaf extract of Tabebuia bahamensis was subjected to bioactivity-directed flash chromatography using silica (40-63 μm particle size and 60 Å porosity) as the stationary phase in a column of length 90 cm (length) × 5 cm (diameter). The elution was carried out using hexane/EtOAc step gradient as
39
illustrated in figure 2.8. Each solvent system was used at a volume of 500 mL to 1l based on the movement of the column by the solvent.
Fig 2.7 Crystals of SF D after toluene slow cooling recrystallization
The different fractions were analyzed with thin layer chromatography (TLC) by spotting few drops of concentrated fraction samples and viewing the TLC plates under
UV light. The fractions with similar retention factor (Rf) value were combined to obtain
SFs. Fractions 51 through 90 formed SF F.
The SFs were then purified by vapor diffusion recrystallization method using
EtOAc and pentane solvents. The samples were dissolved in suitable deuterated solvent and NMR experiments were run to determine the structures.
40
Fig 2.8 Chromatographic separation scheme for Tabebuia bahamensis extract
2.6 IR experiments
The IR experiments of the samples were run using Perkin Elmer FT-IR
Spectrometer.
2.7 NMR experiments
The samples were dissolved in suitable deuterated solvent (7.5 mg in 750 μL) and
NMR experiments were run in Varian INOVA 500 MHz NMR spectrometer. For most of the samples the following experiments were run:
1H spectrum
13C spectrum
Heteronuclear Single Quantum Coherence (HSQC) spectrum
Heteronuclear Multiple Bond Correlation (HMBC) spectrum
Correlation Spectroscopy (COSY) spectrum
41
The NMR spectra obtained were edited and analyzed by using Mestrenova software version 10.0.
2.8 Antimicrobial screening
The isolated compounds were screened for antibacterial activity against Gram- positive bacteria Bacillus cereus, (ATCC No. 14579) and Staphylococcus aureus (ATCC
No. 29213), and Gram-negative bacteria Escherichia coli (ATCC No. 10798). The minimum inhibitory concentrations (MIC) of the compounds against these microbes were determined by microbroth dilution technique as demonstrated by Sahm and Washington,
1991. Exactly 50 μL of 1% w/v solutions of the samples in DMSO was put in a well of
96 well plates and 50 μL of cation–adjusted Mueller Hinton broth (CAMHB) was added.
The sample solutions were then serially diluted (1:1) by transferring 50 μL of sample-
CAMHB mixture to the next lane and adding 50 μL fresh CAMHB to obtain concentration from 2500 μg/mL to 12.5 μg/mL. The microbes were added to each well at a concentration of approximately 1.5 x 108 colony forming units (CFU)/mL. The plates were incubated at 37̊ C for 24 hours and the final MIC was determined as the lowest concentration without any turbidity in the solution. Gentamicin was used as positive antimicrobial control and DMSO was used as negative control.
Antifungal activity of the samples against Candida albicans (ATCC No. 90028) and Aspergillus niger (ATCC No. 16888) were determined as described above, in yeast- nitrogen base growth medium with final concentration of approximately 7.5 x 107
CFU/mL. Amphotericin B was used as positive control and DMSO was used as negative control for antifungal tests.
42
2.9 Cytotoxicity test
The compound F from T. bahamensis was screened for cytotoxic activity against
MCF-7 (breast cancer) using MTT-based Cytotoxicity Assay (Berridge, Herst, and Tan
2005). Human MCF-7 breast adenocarcinoma cells (ATCC No. HTB-22) (Soule et al.
1973) were grown in a 5% CO2 environment at 37°C in RPMI-1640 medium, supplemented with 10% FBS, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, 15mM of HEPES, and buffered with 26.7 mM sodium bicarbonate (NaHCO3) to pH 7.35. Cells were plated into 96-well cell culture plates at concentration of 2.5 ×104 cells/well. The volume in each well was 100 μL. After 48 hours, supernatant fluid was removed by suction and replaced with 100 μL growth medium containing 1.0 μL of
DMSO solution of the sample (1% w/w in DMSO), giving a final concentration of
100μg/mL for each well. Solutions were added to wells in four replicates. Medium controls and DMSO controls (10 μL DMSO/mL) were used. Tingenone was used as a positive control. After the addition of compounds, plates were incubated for 48 h at 37°C in 5% CO2; medium was then removed by suction, and 100 μL of fresh medium was added to each well. In order to establish percent kill rates, the MTT assay for cell viability was carried out (Ferrari, Fornasiero, and Isetta 1990). After colorimetric readings were recorded using SpectraMAX Plus microplate reader, at 570 nm, percent kill was calculated. The results were recorded in Table 3.8.
Cytotoxicity screenings were performed by Noura Dosoky of the Natural Products
Drug Discovery Research group.
43
CHAPTER THREE
RESULTS
3.1 Compounds from Ruyschia phylladenia
The phytochemicals obtained from Ruyschia phylladenia were identified by using
NMR spectra and IR spectra of the samples.
3.1.1 Characterization of compound A
From superfraction A, colorless powder was obtained. Analysis of NMR and IR spectra led to the conclusion that compound A is lupeol.
Fig 3.1 Chemical structure of lupeol
44
Molecular formula: C30H50O
Molecular Weight: 426 g/mol
Yield: 25 mg
Percentage Yield: 0.1%
1 H NMR (500 MHz, CDCl3) δ 4.69 (d, J = 2.1 Hz, 1H), 4.57 (s, 1H), 3.19 (s, 1H), 2.36
(s, 1H), 1.68 (s, 3H), 1.40 – 1.38 (m, 4H), 1.36 (s, 1H), 1.03 (s, 3H), 1.00 (s, 1H), 0.97 (s,
3H), 0.94 (s, 3H), 0.94 (s, 1H), 0.87 (s, 2H), 0.83 (s, 3H), 0.79 (s, 3H), 0.76 (s, 3H).
The protons with chemical shifts of 4.57 and 4.69 ppm represent the olefinic protons (-C=CH2). The proton with chemical shift of 3.19 ppm represents the proton directly attached to C–O carbon. There are seven methyl singlets at δ 0.76, 0.79, 0.83,
0.94, 0.97, 1.03 and 1.68 ppm determined on the basis of integration. The tall peak at
1.26 ppm is an impurity peak of water in CDCl3 (Fulmer et al. 2010). The peak at 7.26 ppm is the solvent peak. The proton spectra are presented in fig 3.3 and 3.4.
13 C NMR (126 MHz, CDCl3) δ 151.13, 109.47, 79.18 , 55.46, 50.60, 48.46, 48.15, 43.16,
42.99, 40.99, 40.16, 39.02, 38.87, 38.22, 37.33, 35.74, 34.44, 30.01, 28.15, 27.61, 27.58,
25.31, 21.09, 19.48, 18.51, 18.16, 16.28, 16.14, 15.53, 14.71.
There are total of 30 carbon peaks of lupane skeleton as seen in carbon spectrum
(fig 3.5, 3.6). The chemical shifts of 151.13 and 109.47 ppm represent the alkenic carbons (-C=C-) while the chemical shift of 79.18 ppm represent the carbon bonded to hydroxyl group (–C-OH). The peak at 77.2 ppm represents the solvent while the longer peak at 29.8 ppm is the water impurity (Fulmer et al. 2010).
45
The HSQC spectra (fig 3.7, 3.8) indicate the presence of 11 methylene (–CH2-) groups at carbon δ 18.51, 21.09, 25.31, 27.58, 27.61, 30.01, 34.44, 35.74, 38.87, 40.16 and 109.47, and the presence of 6 methine (-CH-) groups at carbon δ 38.22, 48.15, 48.46,
50.60, 55.46, and 79.18. The HSQC spectrum also reveals the presence of six quaternary carbons at δ 37.33, 39.02, 40.99, 42.99, 43.16 and 151.13 ppm.
The final connections could be made by HMBC. The H C connections from
HMBC spectra (fig 3.9, 3.10) are mentioned in the figure (fig 3.2) below by the arrows.
Fig 3.2 Key HMBC correlation of compound A
KBr -1 -1 -1 -1 IR Spectra υ max (fig 3.11) : 3326.49 cm , 2940.17 cm , 2851.09 cm , 1640.12 cm ,
1452.45 cm-1, 1380.03 cm-1, 1188.63 cm-1, 1104.88 cm-1, 1034.37 cm-1, 983.66 cm-1,
943.90 cm-1, 880.47 cm-1, 728.25 cm-1
46
The NMR spectra and IR spectra of the sample A agree with the data for lupeol provided in literature (Mahato and Kundu 1994; Ahmad and Rahman 1994a; Haque et al.
2006).
Table 3.1 NMR assignment of Compound A (Lupeol)
Position Carbon δ 13C Proton δ 1H 1 38.87 0.90 2 27.61 1.68 3 79.18 3.19 (s, 1H) 4 39.02 - 5 55.46 0.67 6 18.51 1.38, 1.51 7 34.44 1.38 8 40.99 - 9 50.60 1.28 10 37.33 - 11 21.09 1.20, 1.39 12 25.31 1.64 13 38.22 1.65 14 42.99 - 15 27.58 1.56 16 35.74 1.46, 1.49 17 43.16 - 18 48.46 1.35 19 48.15 2.36 20 151.13 - 21 30.01 1.91 22 40.16 1.17, 1.37 23 28.15 0.97 (s, 3H) 24 15.53 0.76 (s, 3H) 25 16.28 0.83 (s, 3H) 26 16.14 1.03 (s, 3H) 27 14.71 0.94 (s, 3H) 28 18.16 0.79 (s, 3H) 29 109.47 4.69 (d, J = 2.1 Hz, 1H), 4.57 (s, 1H) 30 19.48 1.68 (s, 3H)
47
48
Fig 3.3 Proton spectrum of compound A
49
Fig 3.4 Proton spectrum of compound A (Major peaks)
50
Fig 3.5 Carbon spectrum of compound A
51
Fig 3.6 Carbon spectrum of compound A (closer view)
52
Fig 3.7 HSQC spectrum of compound A
5
3
Fig 3.8 HSQC spectrum of compound A (Closer view)
54
Fig 3.9 HMBC spectrum of compound A
55
Fig 3.10 HMBC spectrum of compound A (closer view)
56
Fig 3.11 IR spectrum of compound A
3.1.2 Characterization of compound B
From superfraction B, colorless solid particles were obtained. Analysis of NMR and IR spectra led to the conclusion that compound B is betulinic acid.
Fig 3.12 Chemical structure of betulinic acid
Molecular Formula: C30H48O3
Molecular weight: 456 g/mol
Yield: 600 mg
%Yield: 2.4%
1 H NMR (500 MHz, CDCl3) δ 4.74 (d, J = 2.2 Hz, 1H), 4.61 (d, J = 2.5 Hz, 1H), 3.19
(dd, J = 11.4, 4.8 Hz, 1H), 3.00 (td, J = 10.7, 4.8 Hz, 1H), 2.27 (dt, J = 13.0, 3.4 Hz, 1H),
2.19 (td, J = 12.3, 3.6 Hz, 1H), 2.04 – 1.93 (m, 2H), 1.69 (s, 3H), 0.98 (s, 3H), 0.97 (s,
3H), 0.93 (s, 3H), 0.83 (s, 3H), 0.75 (s, 3H), 0.70 – 0.66 (m, 1H).
57
The proton spectrum (fig 3.14, 3.15) has signals with chemical shifts of 4.61 and
4.74 ppm that represent the proton attached to olefinic carbon (C=CH2). The peak with chemical shift of 3.19 ppm represents the proton directly attached to carbon containing hydroxyl group (H- C–OH). There are six methyl singlets at δ 0.75, 0.83, 0.93, 0.97,
0.98 and 1.69 ppm. The tall peak at 1.26 ppm is an impurity peak of water in CDCl3
(Fulmer et al. 2010). The peak at 7.26 ppm is the solvent peak.
The NMR of compound B run in DMSO-d6 confirms the presence of a carboxylic acid proton at chemical shift of 12.05 ppm (fig 3.16).
13 C NMR (126 MHz, CDCl3) δ 181.13, 150.55, 109.84, 79.17, 56.50, 55.50, 50.67, 49.43,
47.06, 42.59, 40.85, 39.02, 38.87, 38.57, 37.37, 37.20, 34.48, 32.32, 30.72, 29.86, 28.15,
27.56, 25.66, 21.01, 19.54, 18.44, 16.28, 16.19, 15.50, 14.86.
There are total of 30 carbon peaks in carbon NMR spectrum (fig 3.17, 3.18). The carbons with chemical shift of 150.55 and 109.84 ppm represent the alkenic (–C=C-) carbons while the chemical shift of 79.17 ppm represents carbon bonded to hydroxyl group (–C-OH). The peak at 181.13 ppm indicates the presence of a carboxyl carbon
(C=O). The carbon shift of δ 77.2 ppm represents the solvent carbon.
The blue peaks in HSQC spectra (fig 3.19, 3.20) indicate the presence of
11methylene groups at carbon shifts of 18.44, 21.01, 25.66, 27.56, 29.86, 30.72, 32.32,
34.48, 37.20, 38.87 and 109.84 ppm. HSQC also indicates the presence of 6 methine
(-CH-) groups at carbon shift of 38.57, 47.06, 49.43, 50.67, 55.50, and 79.17 ppm. There are seven quaternary carbons at δ 37.37, 39.02, 40.85, 42.59, 56.50, 150.55 and 181.13 ppm confirmed on the basis of absence of crosspeaks.
58
The final connections could be made by HMBC (fig 3.21, 3.22) and COSY
(fig3.23) spectra. The H C connections from HMBC and H H connections from COSY are mentioned in the figure (fig 3.13) below by the arrows.
Fig 3.13: Key HMBC and COSY correlations of compound B
KBr -1 -1 -1 -1 IR Spectra υ max (fig 3.24): 3418.99 cm , 3074.77 cm , 2938.79 cm , 2867.99 cm ,
1684.98 cm-1, 1642.08 cm-1 1447.58 cm-1, 1375.61cm-1, 1235.68 cm-1, 1032.44 cm-1,
881.95 cm-1.
The NMR spectra and IR spectra of the compound B agree with the data for betulinic acid provided in literature (Ahmad and Rahman 1994b; Mahato and Kundu
1994; Tadesse, Reneela, and Dekebo 2012; Cîntǎ Pînzaru, Leopold, and Kiefer 2002).
59
Table 3.2 NMR assignment of compound B (Betulinic acid)
Position Carbon δ 13C Proton δ 1H 1 38.87 0.90, 1.66 2 27.56 1.61 3 79.17 3.19 (dd, J = 11.4, 4.8 Hz) 4 39.02 - 5 55.50 0.68 6 18.44 1.37, 1.53 7 34.48 1.38 8 40.85 - 9 50.67 1.27 10 37.37 - 11 21.01 1.21 12 25.66 1.04, 1.71 13 38.57 2.19 14 42.59 - 15 30.72 1.41, 1.98 16 32.32 1.42, 2.27 17 56.50 - 18 49.43 1.61 19 47.06 3.00 20 150.55 - 21 29.86 1.19, 1.26 22 37.20 1.49, 1.98 23 28.15 0.97 (s, 3H) 24 15.50 0.75 (s, 3H) 25 16.28 0.83 (s, 3H) 26 16.19 0.93 (s, 3H) 27 14.86 0.98 (s, 3H) 28 181.13 - 29 109.84 4.74 (d, J = 2.2 Hz, 1H), 4.61 (d, J = 2.5 Hz, 1H) 30 19.54 1.69 (s, 3H)
60
61
Fig 3.14 Proton spectrum of compound B in chloroform-d
62
Fig 3.15 Proton spectrum of compound B in chloroform-d (Major Peaks)
63
Fig 3.16 Proton spectrum of compound B in DMSO-d6
64
Fig 3.17 Carbon spectrum of compound B in chloroform-d
65
Fig 3.18 Carbon spectrum of compound B (closer view)
66
Fig 3.19 HSQC spectrum of compound B
67
Fig 3.20 HSQC spectrum of compound B (closer view)
68
Fig 3.21 HMBC spectrum of compound B
69
Fig 3.22 HMBC spectrum of compound B (closer view)
70
Fig 3.23 COSY spectrum of compound B
71
Fig 3.24 IR spectrum of compound B
3.2 Compounds from Eugenia monteverdensis
The phytochemicals obtained from Eugenia monteverdensis were identified by using NMR spectra of the samples.
3.2.1 Characterization of compound C
From superfraction C, colorless, long crystals were obtained. Analysis of NMR spectra led to the conclusion that compound C is β-Sitosterol.
Fig 3.25 Chemical structure of β-Sitosterol
Molecular formula: C29H50O
Molecular weight: 414.71 g/mol
Yield: 180 mg
%Yield: 0.72%
1H NMR (500 MHz, Chloroform-d): δ 5.35 (t, J = 5.3, 2.0 Hz, 1H), 3.52 (tt, J = 11.2, 4.7
Hz, 1H), 1.01 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 1.8 Hz, 3H), 0.82 (d, J = 4.1
Hz, 3H), 0.81 (s, 3H), 0.68 (s, 3H).
72
The proton spectrum is presented in fig 3.27 and fig 3.28. The single proton at
5.35 ppm represents the olefinic proton. The single proton at chemical shift of 3.52 ppm corresponds to proton directly connected to –C-O carbon. There are six methyl groups at chemical shifts of 1.01, 0.92, 0.84, 0.82, 0.81, 0.68 ppm. The chloroform solvent peak is at 7.26 ppm.
13C NMR (126 MHz, Chloroform-d) δ 140.90, 121.87, 71.97, 56.92, 56.21, 50.29, 46.00,
42.48, 42.46, 39.93, 37.41, 36.67, 36.30, 34.11, 32.07, 32.07, 31.83, 29.31, 28.41, 26.23,
24.46, 23.23, 21.24, 19.98, 19.56, 19.19, 18.94, 12.14, 12.02.
The carbon spectrum (fig 3.29, 3.30) indicates the presence of 29 carbons. The carbon peaks at chemical shifts of 140.90 and 121.87 ppm correspond to the double bonded (–C=C-) carbons. The carbon peak at 71.97 ppm indicates the carbon attached to alcohol group (C-OH). All other carbons are saturated. The solvent peak is at 77.2 ppm.
The HSQC spectrum (fig 3.31, 3.32) indicates the presence of 11 methylene (–
CH2-) groups at carbon δ 21.24, 23.23, 24.46, 26.23, 28.41, 31.83, 32.07, 34.11, 37.41,
39.93, 42.46 ppm. The crosspeak at carbon shift of 29.86 ppm was determined to be an impurity based on the absence of HMBC crosspeaks of that particular carbon. HSQC also indicates the presence of 9 methine (-CH-) groups at carbon δ 29.31, 32.07, 36.30,
46.00, 50.29, 56.21, 56.92, 71.97 and 121.87 ppm. The HSQC spectrum specifies the presence of three quaternary carbons at carbon δ 36.67 and 42.48 140.90 ppm. The six methyl groups correspond to carbon at 12.02, 12.14, 18.94, 19.19, 19.56, and19.98 ppm.
73
The final connections could be made by HMBC (fig 3.33, 3.34) and COSY (fig 3.35).
The H C connections from HMBC and H H connections from COSY are mentioned in the figure (fig 3.26) below by the arrows.
Fig 3.26 Key HMBC and COSY correlations of compound C
The NMR spectra obtained for sample C agree with the NMR data for β-Sitosterol provided in literature (Chaturvedula and Prakash 2012; Habib et al. 2007).
74
Table 3.3 NMR assignment of compound C (β-Sitosterol)
Position Carbon δ 13C Proton δ 1H 1 37.41 1.07, 1.85 2 31.83 1.51, 1.83 3 71.97 3.52 (tt, J = 11.2, 4.7 Hz, 1H) 4 42.46 2.25 5 140.90 - 6 121.87 5.35 (t, J = 5.3, 2.0 Hz, 1H) 7 32.07 1.95, 1.98 8 32.07 1.44 9 50.29 0.93 10 36.67 - 11 21.24 1.45-1.49 12 39.93 1.15, 2.01 13 42.48 - 14 56.92 0.98 15 26.23 1.16 16 28.41 1.84 17 56.21 1.11 18 36.30 1.35 19 19.19 0.92 (d, J = 6.5 Hz, 3H) 20 34.11 1.32 21 24.46 1.57 22 46.00 0.94 23 23.23 1.23-1.26 24 12.14 0.84 (d, J = 1.8 Hz, 3H) 25 29.31 1.66 26 19.98 0.82 (d, J = 4.1 Hz, 3H) 27 19.56 0.81 (s, 3H) 28 19.19 0.68 (s, 3H) 29 12.02 1.01 (s, 3H)
75
76
Fig 3.27 Proton spectrum of compound C
77
Fig 3.28 Proton spectrum of compound C (Major peaks)
78
Fig 3.29 Carbon spectrum of compound C
79
Fig 3.30 Carbon spectrum of compound C (closer view)
80
Fig 3.31 HSQC spectrum of compound C
81
Fig 3.32 HSQC spectrum of compound C (closer view)
82
Fig 3.33 HMBC spectrum of compound C
83
Fig 3.34 HMBC spectrum of compound C (closer view)
84
Fig 3.35 COSY spectrum of compound C
3.2.2 Characterization of compound D
From superfraction D, colorless solid particles were obtained. The proton and carbon spectra (fig 3.36 and 3.37 respectively) of this compound matched exactly with that of compound B. Hence it could be easily concluded that compound D is betulinic acid.
Molecular Formula: C30H48O3
Molecular weight: 456 g/mol
Yield:152 mg
%Yield: 0.6%
85
86
Fig 3.36 Proton spectrum of compound D
87
Fig 3.37 Carbon spectrum of compound D
3.2.3 Characterization of compound E
From superfraction E colorless solid needles were obtained. Analysis of NMR spectra led to the conclusion that compound E is barbinervic acid.
Fig 3.38 Chemical structure of barbinervic acid
Molecular formula: C30H48O5
Molecular weight: 488.699 g/mol
Yield: 460 mg
% Yield: 1.84%
1 H NMR (500 MHz, DMSO-d6) δ 11.84 (s, 1H), 5.16 (m, 1H), 4.11 (t, J = 5.3 Hz, 1H),
4.07 (d, J = 4.6 Hz, 1H), 3.77 (d, J = 1.1 Hz, 1H), 3.57 (dt, J = 5.0, 2.7 Hz, 1H), 3.44 (dd,
J = 10.9, 5.3 Hz, 1H), 3.21 (dd, J = 10.9, 5.2 Hz, 1H), 2.36 (s, 1H), 1.30 (s, 3H), 1.28 (s,
1H), 1.08 (s, 3H), 0.89 (s, 3H), 0.84 (d, J=10 Hz 3H), 0.82 (s, 3H), 0.67 (s, 3H).
88
The proton spectrum is presented in fig 3.40 and 3.41. The signal at 11.84 ppm represents carboxylic acid proton and that at 5.16 ppm represents the proton attached to double bonded carbon. The doublet peaks at chemical shifts of 4.11, 4.07 and 3.77 ppm corresponds to hydroxyl (O-H) protons. The peaks at 3.44 and 3.21 belong to methylene protons attached to hydroxyl group. There are six methyl groups at chemical shifts of
1.30, 1.08, 0.89, 0.84, 0.82, 0.67 ppm. The solvent peak is at 2.50 ppm.
13 C NMR (126 MHz, dmso-d6) δ 178.92, 138.52, 126.87, 71.61, 68.35, 64.12, 53.17,
48.89, 46.88, 46.61, 42.45, 41.39, 41.08, 39.46, 37.26, 36.45, 33.07, 32.86, 27.99, 26.41,
25.93, 25.17,25.16, 24.00, 23.25, 22.69, 18.17, 16.45, 16.29, 15.32.
The carbon spectrum (fig 3.42, 3.43) indicates the presence of 30 carbons. The carbon peaks at chemical shifts of 178.92 ppm indicates a carboxylic acid and peaks at
138.52 and 126.87 ppm correspond to the alkenic carbons (–C=C-). The signals at 71.61,
68.35 and 64.12 ppm indicate the carbons attached to alcohol group (C-OH). All other carbons are saturated. The solvent peak is at 39.5 ppm.
The HSQC (fig 3.44, 3.45) indicated the presence of 9 methylene (–CH2-) groups at carbon shifts δ 18.17, 23.25, 25.16, 25.17, 25.93, 27.99, 32.86, 33.07 and 37.26 ppm.
HSQC also reveals the presence of 6 methine carbons at chemical shifts of 41.39, 46.61,
48.89, 53.17, 68.35 and 126.87 ppm. The HSQC spectrum specified the presence of 7 quaternary carbons at chemical shifts of 36.45, 39.46, 41.08, 42.45, 46.88, 71.61 and
138.52 ppm. The final connections could be made by HMBC (fig 3.46, 3.47) and COSY
(fig 3.48). The H C connections from HMBC and H H connections from
COSY are mentioned in the figure 3.39 below by the arrows.
89
Fig 3.39 Key HMBC and COSY correlations of compound E
The NMR spectra obtained for sample E agree with the NMR data for barbinervic acid provided in literature. (J.-P. Fan and He 2006; Takani et al. 1977; K. Takahashi and
Takani 1978).
90
Table 3.4 NMR assignment of compound E (Barbinervic acid)
Position Carbon δ 13C Proton δ 1H 1 32.86 1.28, 1.32 2 25.16 1.37 3 68.35 3.57 (dt, J = 5.0, 2.7 Hz, 1H) 4 42.45 - 5 48.89 1.28 6 18.17 1.28, 1.40 7 33.07 1.19, 1.42 8 39.46 - 9 46.61 1.69 10 36.45 - 11 23.25 1.82, 1.93 12 126.87 5.16 (m, 1H) 13 138.52 - 14 41.08 - 15 27.99 0.90, 1.67 16 25.17 1.77, 2.49 17 46.88 - 18 53.17 2.36 19 71.61 - 20 41.39 1.25 21 25.93 1.14, 1.63 22 37.26 1.51, 1.59 23 22.69 0.89 (s, 3H) 24 64.12 3.44 (dd, J = 10.9, 5.3 Hz, 1H), 3.21 (dd, J = 10.9, 5.2 Hz, 1H) 25 15.32 0.82 (s, 3H) 26 16.45 0.67 (s, 3H) 27 24.00 1.30 (s, 3H) 28 178.92 29 26.41 1.08 (s, 3H) 30 16.29 0.84 (d, J=10 Hz 3H)
91
92
Fig 3.40 Proton spectrum of compound E
93
Fig 3.41 Proton spectrum of compound E (Major peaks)
94
Fig 3.42 Carbon spectrum of compound E
95
Fig 3.43 Carbon spectrum of compound E (closer view)
96
Fig 3.44 HSQC spectrum of compound E
97
Fig 3.45 HSQC spectrum of compound E (closer view)
98
Fig 3.46 HMBC spectrum of compound E
99
Fig 3.47 HMBC spectrum of compound E (closer view)
100
Fig 3.48 COSY spectrum of compound E
3.3 Compounds from Tabebuia bahamensis
The phytochemical obtained from Tabebuia bahamensis was identified by using
NMR spectra of the samples.
3.3.1 Characterization of compound F
From superfraction F, white powder was obtained. Analysis of NMR spectra led to the conclusion that compound F is ursolic acid.
Fig 3.49 Chemical structure of ursolic acid
Molecular Formula: C30H48O3
Molecular weight: 456 g/mol
Yield: 6.1 gm
%Yield: 24.4% 101
1 H NMR (500 MHz, DMSO-d6) δ 11.93 (s, 1H), 5.13 (t, J = 3.7 Hz, 1H), 4.29 (d, J = 5.1
Hz, 1H), 3.00 (dt, J = 10.1, 5.0 Hz, 1H), 2.54 (s, 1H), 2.10 (dd, J = 11.4, 1.6 Hz, 1H),
1.04 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.81 (d, J = 6.4 Hz, 3H), 0.75 (s,
3H), 0.67 (s, 3H).
The proton spectrum is presented in fig 3.51 and 3.52. The peak at 11.93 ppm represents carboxylic acid proton and that at 5.13 ppm represents the proton attached to double bonded carbon. The doublet peak at chemical shift of 4.29 ppm corresponds to alcoholic (O-H) proton. There are seven methyl groups at chemical shifts of 1.04, 0.91,
0.89, 0.86, 0.81, 0.75 and 0.67 ppm. The solvent peak is at 2.50 ppm.
13 C NMR (126 MHz, dmso-d6) δ 178.24, 138.17, 124.55, 76.81, 54.76, 52.36, 47.00,
46.81, 41.63, 39.09, 38.49, 38.42, 38.37, 38.21, 36.52, 36.30, 32.69, 30.17, 28.25, 27.53,
26.98, 23.79, 23.26, 22.84, 21.07, 17.99, 17.01, 16.91, 16.07, 15.22.
The carbon spectrum (fig 3.52, 3.54) indicates the presence of 30 carbons. The carbon peak at chemical shift of 178.24 ppm indicates a carboxylic acid and peaks at
138.17 and 124.55 ppm correspond to the alkenic carbons (–C=C-). The peak at 76.52 ppm indicates the carbon attached to alcohol group (C-OH). All other carbons are saturated. The solvent peak is at 39.5 ppm.
The HSQC spectrum (fig 3.55, 3.56) indicates the presence of 9 methylene (–
CH2-) groups at carbon shifts of 17.99, 22.84, 23.79, 26.98, 27.53, 30.17, 32.69, 36.30 and 38.21 ppm. HSQC also reveals the presence of 7 methine carbons at chemical shifts of 38.42, 38.49, 47.00, 52.36, 54.76, 76.81 and 124.55 ppm. The HSQC spectra specified
102
the presence of 7 quaternary carbons at chemical shifts of 39.09, 36.52, 38.37, 41.63,
46.81, 138.17 and 178.24 ppm.
The final connections could be made by HMBC (fig 3.57, 3.58). The H C connections from HMBC are mentioned in the figure (fig 3.50) below by the arrows.
Fig 3.50 Key HMBC correlations of compound F
103
Table 3.5 NMR assignment of compound F (Ursolic Acid)
Position Carbon δ 13C Proton δ 1H 1 36.30 1.56 2 26.98 1.44 3 76.81 3.00 (dt, J = 10.1, 5.0 Hz, 1H) 4 38.37 - 5 54.76 0.67 6 17.99 1.30, 1.47 7 32.69 1.27 8 39.09 - 9 47.00 1.47 10 36.52 - 11 22.84 1.85 12 124.55 5.13 (t, J = 3.7 Hz, 1H) 13 138.17 - 14 41.63 - 15 27.53 1.00, 1.79 16 23.79 1.52, 1.93 17 46.81 - 18 52.36 2.11 19 38.49 0.92 20 38.42 1.30 21 30.16 1.27, 1.42 22 38.22 1.53 23 28.25 0.89 (s, 3H) 24 16.07 0.67 (s, 3H) 25 15.22 0.86 (s, 3H) 26 16.91 0.75 (s, 3H) 27 23.26 1.04 (s, 3H) 28 178.24 - 29 17.01 0.81 (d, J = 6.4 Hz, 3H) 30 21.07 0.91 (s, 3H)
104
105
Fig 3.51 Proton spectrum of compound F
106
Fig 3.52 Proton spectrum of compound F (Major Peaks)
107
Fig 3.53 Carbon spectrum of compound F
108
Fig 3.54 Carbon Spectrum of compound F (closer view)
109
Fig 3.55 HSQC spectrum of compound F
110
Fig 3.56 HSQC spectrum of compound F (closer view)
111
Fig 3.57 HMBC spectrum of compound F
112
Fig 3.58 HMBC spectrum of compound F (closer view)
3.4 Result of antileishmanial assay
Table 3.6 Antileishmanial activity (μg/mL) of Betulinic acid
Compound IC50 against L. amazonensis CC50 against mouse promastigotes macrophage cells Betulinic acid 11.3±4.9 μg/mL 14.1±4.7 μg/mL
3.5 Results of antimicrobial tests
Table 3.7 Antimicrobial activity (IC50 in μg/mL) of sample compounds
Compound Lupeol Betulinic β-Sitosterol Barbinervic Ursolic Acid acid Acid
Microbe E. coli >2500 >2500 312.5 312.5 >2500
B. cereus >2500 >2500 312.5 156 >2500
S. aureus >2500 >2500 156 >2500 156
C. albicans >2500 >2500 312.5 >2500 >2500
A. niger >2500 >2500 39 >2500 >2500
3.6 Results of cytotoxicity assay
Table 3.8 MTT Cytotoxicity Assay result
Compound Percentage Kill Std. Deviation of % kill Ursolic acid 100.13 0.50
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CHAPTER FOUR
DISCUSSION
Lupeol (Lup-20(29)-en-3β-ol), also known as clerodol, fagasterol and lupenol is a common triterpenoid found in various plants like white cabbage, pepper, cucumber, tomato, and fruits like strawberry, red grapes and mango. It has importance in pharmacological areas as it has shown anti-inflammatory, anti-arthritic and anti-cancer activities. Lupeol has also been found to be beneficial against diabetes, heart disease, renal toxicity and hepatic toxicity (Wal et al. 2011). Geeta et al. have reported the successful use of lupeol against arthritis in mouse (Geetha and Varalakshmi 1999). It has also been reported to exhibit strong anti-mutagenic activity under in vivo and in vitro conditions (Walclecio et al. 2008; Nigam, Prasad, and Shukla 2007). Wal et al. have demonstrated that lupeol inhibits the growth of highly metastatic tumors of human melanoma origin in vitro and in vivo (Wal et al. 2011).
Lupeol has been reported to be active against several protozoa that cause malaria, trypanosomiasis and leishmaniasis. Fournet et al. have studied the activity of lupeol extracted from Pera benensis against several species of Leishmania and found that lupeol inhibits them significantly. This compound has shown IC90 value of 100μg/mL against
114
promastigotes and amastigotes of 5 species of Leishmania including L. braziliensis, L. amazonensis and L. donovani (Fournet et al. 1992; Wal et al. 2011; Siddique and Saleem
2011). Ogungbe et al. performed a docking study of lupeol with several target sites of different Leishmania spp. and found that it docked very effectively with nucleoside diphosphate kinase B (NDKb), tyrosyl-tRNA synthetase and oligopeptidase B of L. major; glycerol-3-phosphate dehydrogenase, pyruvate kinase of L. mexicana; cathepsin B of L. donovani and sterol 14-α demethylase of L. infantum , indicating these enzymes as probable target sites (Ogungbe and Setzer 2013).
Lupeol caffeate from Betula ermanii Cham. has shown activity against L. major with IC50>100 μg/mL (M. Takahashi et al. 2004). One major advantage of lupeol is that its toxicity is very low. Even at the oral dosage of 2g/kg, lupeol produced no adverse effects in rats and mice and no mortality was recorded even after 96 hours (Patočka
2003). Lupeol extracted from leaves of Morinda morindoides has shown antiplasmodial activity (Tolstikova et al. 2006; Tona et al. 2004).
On the basis of antimicrobial tests performed as part of this study, lupeol was found to be inactive against E. coli, B. cereus and S. aureus. which corresponds to the study by Mathabe et al. (Mathabe et al. 2008; Gallo and Sarachine 2009). It was also found to be inactive against Candida albicans and Aspergillus niger from the tests performed. Inactivity of lupeol as antifungal has been reported in literature (Gallo and
Sarachine 2009).
Betulinic acid is a common natural triterpene that have been isolated from various plants including Quisqualis fructus, Coussarea paniculata, Caesalpinia paraguariensis,
Vitex negundo, Ilex macropoda, Anemone raddeana and Dolicarpus schottianus.
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Betulinic acid is a compound of pharmacological importance because of its various medicinal activities. It has been reported to inhibit HIV protease (Xu et al. 1996), reverse transcriptase, entry of HIV-1 (G. Li et al. 2010) and inhibits HIV replication (Mayaux et al. 1994; Patočka 2003; W. N. Setzer et al. 2000). It has also shown activity against human melanoma, neuroectodermal and malignant tumor cells. Selzer et al. studied the growth-inhibitory properties of this compound and concluded that it strongly and consistently suppresses the growth and colony-forming ability of all human melanoma cells investigated, while the activity was less pronounced in human melanocytes (Selzer et al. 2000). Zuco et al., Fulda et al., Jeong et al. are some groups who have identified betulinic acid as a significant anticancer agent (Yogeeswari and Sriram 2005). In the study of over 3000 plant extracts investigated for antitumor activity by National Cancer
Institute (NCI), USA, betulinic acid has stood out as potential anticancer agent (Patočka
2003). It has also shown anti-inflammatory activity, antihelminthic activity against
Caenorhabditis elegans (Yogeeswari and Sriram 2005) and antimalarial activity (Paduch et al. 2007).
In the current study, betulinic acid was found to be very effective against promastigotes of L. amazonensis. Unfortunately, it also inhibited mouse macrophages in a significant manner, discouraging its use as antileishmanial product against L. amazonensis. There have been few other studies of antileishmanial activity of betulinic acid. Takahashi et al. studied the antileishmanial activity of betulinic acid obtained from
Betula platyphylla var. japonica and determined the IC50 to be 40±3.2 μg/mL against L. major (M. Takahashi et al. 2004). Alakurtti et al. have reported betulinic acid to possess moderate anti-leishmanial activity against L. major with 40% inhibition at 50 μM
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concentration (Alakurtti et al. 2010). The probable site of action in L. major may be tyrosyl-tRNA synthetase, N- myristoyltransferase or nucleoside hydrolase as determined by docking study performed by Ogungbe et al (Ogungbe and Setzer 2013). Conversely,
Dominguez –Carmona et al. have reported betulinic acid to have no leishmanicidal activity against L. amazonensis and L. braziliensis. However, betulinic acid has shown strong antiplasmodial activity against chloroquine-resistant P. falciparum parasites in vitro, with IC50 of 9.9 μM (Ghaffari Moghaddam, Ahmad, and Samzadeh-Kermani 2012).
Derivatives of betulinic acid are reported to be active against several species of
Leishmania. Betulin aldehyde has displayed anti-leishmanial activity with 64% inhibition at 50 μM concentration compared to betulinic acid where as betulonic acid inhibited 98% of L. donovani at 50 μM. Dihydrobetulinic acid has shown to cause apoptosis in L. donovani with IC50 of 2.6 μM and 4.1 μM against promastigotes and amastigotes
(Alakurtti et al. 2010).
Betulinic acid was tested against E. coli, B. cereus S. aureus, C. albicans and A. niger for its biological activity. However it was found to be active against none. This lack of activity against these microorganisms has also been reported by Wldemichael et al. (Woldemichael et al. 2003).
β-Sitosterol (24α-ethylcholesterol) is one of the most predominant phytosterols found commonly in corn, cottonseed, peanut and linseed oils. Phytosterols resemble cholesterol in function and structure but phytosterol side chains contain 9 or 10 carbon atoms unlike cholesterol that has 8 carbon side chain. These compounds are not synthesized in mammals and are derived from plant sources. β-Sitosterol has a wide range of medicinal activities. Prieto et al. and Loizou et al. have reported the anti-
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inflammatory activity of β-Sitosterol (Saeidnia et al. 2014). It has been shown to exhibit anti-inflammatory activity in human aortic endothelial cells, as reported by Loizou et al.
(Loizou et al. 2010). Ovesna et al. have studied β-Sitosterol against tumor cells and revealed that the compound can affect different levels of tumor development (Ovesná,
Vachálková, and Horváthová 2004). β-Sitosterol can inhibit the proliferation of MCF-7 cells in dose dependent manner (Jw, Ur, and Ms 2008). Wilt et al. concluded that β-
Sitosterol could improve the urinary symptoms and flow in patients suffering from
Benign Prostatic Hyperplasia (BPH) (Saeidnia et al. 2014; Lowe and Ku 1996).
Moreover, β-Sitosterol does not have toxic effects in human and animals (Lowe and Ku
1996; Kritchevsky and Chen 2005).
β-Sitosterol-3-O-β-D-glucopyranoside, a derivative of β-Sitosterol, has shown moderate activity against Entamoeba histolytica and Giardia lamblia (Calzada et al.
2003). In silico study of β-Sitosterol against several target sites of L. major indicates methionyl-tRNA synthetase, phosphodiesterase 1and uridine diphosphate-glucose phosphorylase as major target sites among many. The target site in L. Mexicana can be phosphomannomutase or pyruvate kinase while it can be cyclophilin or cathepsin B in L. donovani and 14α-demethylase in L. infantum (Ogungbe and Setzer 2013).
The biological activity of β-Sitosterol against E. coli, B. cereus and S. aureus agrees with the study by Beltrame et al. who have mentioned β-Sitosterol to have MIC of
> 100 μg/mL against S. aureus and against E. coli (Beltrame et al. 2002). In the current study, β-Sitosterol was found to be very active against A. niger with MIC of 39 μg/mL.
Similar activity of this compound against A. niger has been reported by Aderiye et al.
(Aderiye et al. 1989).
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Barbinervic acid has shown to cause reduction in vascular tonus in rat renal circulation and also in rat thoracic aorta (Araújo de Brito et al. 2013). Barbinervic acid is one of the main components of Diospyros kaki which is a traditional Chinese medicine used for the treatment of stroke as hypotensive drug (J. P. Fan and He 2006; J.-P. Fan and
He 2006). In the current study, barbinervic acid was found to be moderately active against E. coli, and significantly active against B. cereus.
Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a triterpenoid that is common in plants like Calluna vulgaris, Melaleuca leucadendron, Ocimum sanctum, Pyrola rotundifolia among others. Several of these plants with ursolic acid as major constituent are considered medicinal plant for their biological activities. Ursolic acid is active hepatoprotective component of various plants like Solanum incanum, Eucalyptus hubrid,
Sambucus chinesis, and is effective against both acute chemically induced liver injury and chronic liver fibrosis and cirrhosis (Liu 1995; Liu 2005). Ursolic acid is also used for its medicinal purpose as an anti-inflammatory agent and acts by inhibiting histamine release from mast cells and also by inhibiting lipoxygenase and cyclooxygenase activity
(Liu 1995; Ikeda, Murakami, and Ohigashi 2008). It also holds pharmacological importance as anti-hyperlipidemic and antioxidant (Liu 1995; Somova et al. 2003).
The strong cytotoxic effect of ursolic acid is supported by studies performed by various groups of researchers. It has shown to act at various stages of tumor development including tumor initiation and tumor promotion (Ovesná et al. 2004). It has been known to induce apoptosis in tumor cells along with preventing malignant transformation of normal cells (Novotný, Vachálková, and Biggs 2001). Ursolic acid is more potent tumorigenic inhibitor than its isomer oleanolic acid. Study has revealed that the cytotoxic
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effect of this compound is because of its ability to block the cell cycle progression in the
G1 phase and triggering the apoptosis (Ma et al. 2005). As a result of its antiproliferative and apoptotic activities, ursolic acid has potential to be used for treatment of hormone refractory and androgen-sensitive prostate cancer (Kassi et al. 2007).
Peixoto et al. studied the activity of ursolic acid obtained from Miconia langsdorffii against L. amazonensis and found IC50 to be 360.3 μM. This IC50 was lower than that of its isomer oleanolic acid (IC50 439.5 μM). However, the combination of two showed synergistic effect with IC50 of 199.6 μg/mL (Peixoto et al. 2011; Bero et al.
2011). Ursolic acid has been reported to show activity against promastigotes of L. amazonensis at IC50 of 43.8 μg/mL and against promastigotes of L. donovani at IC50 of
3.5μg/mL (Adebayo, Suleman, and Samson 2013; Torres-Santos et al. 2004; Gnoatto et al. 2008). Based on molecular docking study, the most probable target site of this compound can be N-myristoyltransferase in L. major, glycerol-3-phosphate dehydrogenase in L. mexicana, and 14α-demethylase in L. infantum (Ogungbe and Setzer
2013).
Ursolic acid showed no activity against E. coli, B. cereus, C. albicans and A. niger but was active against S. aureus. Wolska et al. and Fontanay et al. have also reported activity of ursolic acid against S. aureus (Wolska et al. 2010; Fontanay et al.
2008).
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CHAPTER FIVE
CONCLUSION
Natural products hold the key to cure against several diseases. The plants obtained from Monte Verde and Abaco Island have the potential to be developed into medicines against several diseases including leishmaniasis and cancer. Lupeol obtained from Ruyschia phylladenia is active against several species of Leishmania. Betulinic acid, on the other hand, might be only moderately active against Leishmania spp., but it shows strong activity against HIV infection and several cancer cells. Moreover, the high yield of betulinic acid from R. phylladenia reveals it as a very good source of this medicinally important compound.
Eugenia monteverdensis also possesses pharmacologically active compounds like β-
Sitosterol, betulinic acid and barbinervic acid. Both β-Sitosterol and barbinervic acid shows activities against several microbes emphasizing the medicinal importance of this plant.
Ursolic acid, the major component of T. bahamensis, is a moderate leishmanicidal but a strong cytotoxic agent against several cancer cell lines, and is considered a good antitumor agent. The yield of ursolic acid from T. bahamensis in such huge amount presents the plant as a major source of this compound with wide arrays of therapeutic importance. It has to be noted that T. bahamensis was extracted with DCM while other two plants were extracted
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with acetone. The polarity of solvent used for extraction may be the reason for different yields of compounds from the plants. Acetone, being more polar, extracts polar compounds which adhere more to silica, leading to lesser yield after separation while DCM extracts less polar compounds leading to more yield after separation.
Plants have been used since ancient times for medicinal purpose. With huge number of plants still to be studied for their therapeutic importance, we can hope that there lies the cure to all the diseases known to mankind, in the wild. Tropical rainforests like that in
Monteverde and Bahamas are treasures of such precious plants. We should explore these natural sources of medicines in quest for elixir of life.
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