Chapter 1
INTRODUCTION
1.1
Medicines from plants Until the beginning of the twentieth century, virtually all medicines were derived
from natural sources, most often plants. Even today, at the start of the twenty-first century, about 80% of the world’s population still use plants and plant extracts as their primary source of drugs and medicinal agents. In the United States, approximately 25% of important prescription drugs are still based on plant-derived compounds. Examples of plant-based drugs are shown in Figure 1.1. The plants from which some of these compounds are obtained are pictured in Figures 1.2 through 1.7. Higher plants not only serve as sources for new drugs, but phytochemicals are also extremely useful as lead structures for synthetic modification leading to optimization of biological activity. Examples of such semisynthetic compounds include etoposide and topotecan (Figure 1.1), which are derivatives of podophyllotoxin and camptothecin, respectively (Figure 1.8).
CH3 O O
O
NH
CH 3
OH
O
N CH 3
OH
H O
O
O
OH
O
O
O
O O
OH
OH
O
O
O
O
N O
Paclitaxel Taxol Anticancer diterpenoid isolated from the Pacific yew tree (Taxus brevifolia, Figure 1.2)
CH3O
OCH3 OH
OH
Etoposide – Semisynthetic antineoplastic agent derived from podophyllotoxin
Topotecan – Semisynthetic antitumor agent based on camptothecin
CH2CH3
H N H
N H CH3O
CH2CH3 OCOCH3
O
OCH3 N
CH3O
O
H
OH N
O N
O
O
HO
O
CH3
N
HO
O O
CH 3 O H
H
CH 3O
OH O
Vinblastine – Antitumor alkaloid from the rosy periwinkle (Catharanthus roseus, Figure 1.7)
CH 3
H H CH 3
O O
N
Quinine – Antimalarial alkaloid from the bark of Cinchona spp. (Figure 1.3)
Artemisinin – Antimalarial sesquiterpene lactone from Artemisia annua
O
O
O
CH 3O
H
CH3 O CH2 OH
O
N H
OH
H OH
H
CH 3O CH3O
OCH3 OH
OH OH
Digitalin – Cardiotonic glycoside from the purple foxglove (Digitalis purpurea, Figure 1.4)
OCH 3
Colchicine – Antigout alkaloid from Colchicum autumnale (Figure 1.5)
Figure 1.1 Plant-derived medicinal agents
OCH3 H
N CH3O
O
O O HO
CH 3
N H
O OCH3 O
H
OCH3
H CH3O
OCH3 O
Reserpine – Antihypertensive alkaloid from Rauwolfia serpentina
Figure 1.2 Taxus brevifolia (Moore, 1998)
Figure 1.3 Cinchona officinalis (Moore, 1998)
Figure 1.4 Digitalis purpurea (Wallace, 1991)
Figure 1.5 Colchicum autumnale (Moore, 1998)
Figure 1.6 Podophyllum peltatum (Moore, 1998)
Figure 1.7 Catharanthus roseus (Wallace, 1991)
OH O O
O O O
N N O
CH3O
OCH3
OH
O
OH
Podophyllotoxin – Antiviral constituent of the mayapple (Podophyllum peltatum, Figure 1.6)
Camptothecin – Antitumor alkaloid (Topoisomerase I inhibitor) from Camptotheca acuminata
Figure 1.8 Structures of lead compounds podophyllotoxin and camptothecin
Often plant-based prescription drugs are discovered and developed because the plant was used in the traditional medicine of some culture (Kinghorn and Balandrin, 1993). Indigenous peoples of the world, using trial and error techniques over hundreds of generations, have discovered myriads of plant-based treatments for various ailments and afflictions. Some important plant species, their families, and traditional medicinal uses are summarized in Table 1.1. In this project, it was not possible to consult an Aboriginal healer for ethnobotanical direction; the Paluma rainforest is in a region not historically inhabited by Aborigines. Based on indirect ethnopharmacological considerations (Table 1.1), this work has concentrated on many of these families and genera. In addition, plant species in this thesis were chosen based on field observations including factors such as lack of herbivory or fungal infestation, as well as physical characteristics such as odor, color, or exudate.
Table 1.1 Ethnobotanical applications of plants Plant
Family
Ethnobotanical Use
Location
Ref.
Acalypha hispida
Euphorbiaceae
Dysentery
Northern Thailand
a
Albizia lebbeck
Mimosaceae
Antiprotozoal
Southeastern Africa
b
Alchornea cordifolia
Euphorbiaceae
Skin infections
Tropical West Africa
b
Alphitonia excelsa
Rhamnaceae
Skin infections
Australia
c
Alstonia scholaris
Apocynaceae
Panacea
India, Indonesia
d
Ampelozizyphus amazonicus
Rhamnaceae
Skin infections
Eastern Amazonia
e
Archidendron clypearia
Mimosaceae
Food poisoning
Northern Thailand
a
Beilschmiedia appendiculata
Lauraceae
Skin infections
China
f
Cassytha filiformis
Lauraceae
Venereal diseases
Tropical West Africa
b
Chamaesyce hyssopifolia
Euphorbiaceae
Antifungal
Western Amazonia
g
Croton dichogamus
Euphorbiaceae
Antiviral
Tropical West Africa
b
Croton jatrophoides
Euphorbiaceae
Antimicrobial, antiviral
Tropical West Africa
b
Croton oblongifolius
Euphorbiaceae
Food poisoning
Northern Thailand
a
Table 1.1 (cont’d) Plant
Family
Ethnobotanical Use
Location
Ref.
Croton riviniaefolius
Euphorbiaceae
Skin diseases
Western Amazonia
g
Cryptocarya aromatica
Lauraceae
Antipyretic
Papua New Guinea
d
Cryptocarya medicinelis
Lauraceae
Respiratory ailments
Solomon Islands
d
Dryandra cordata
Proteaceae
Skin infections, parasites
China
f
Drypetes veriabilis
Euphorbiaceae
Tonic
Eastern Amazonia
e
Elaeocarpus grandiflorus
Elaeocarpaceae
Skin infections
Malaya
d
Elaeocarpus oblongus
Elaeocarpaceae
Antibacterial
India
d
Eucalyptus drepanophylla
Myrtaceae
Skin infections
Australia
h
Eucalyptus globulus
Myrtaceae
Respiratory ailments
Australia
h
Eucalyptus gummifera
Myrtaceae
Venereal diseases, antifungal
Australia
h
Euodia cuculata
Rutaceae
Antifungal
Fiji
d
Euodia glomerata
Rutaceae
Antimalarial
Northern Thailand
a
Euphorbia humifusa
Euphorbiaceae
Dysentery
China
f
Table 1.1 (cont’d) Plant
Family
Ethnobotanical Use
Location
Ref.
Fagara chalydea
Rutaceae
Skin infections
Tropical West Africa
b
Faurea saligna
Proteaceae
Diarrhea
Tropical West Africa
b
Ficus fistulosa
Moraceae
Venereal diseases
Northern Thailand
a
Ficus leprieuril
Moraceae
Antiviral
Tropical West Africa
b
Ficus macrosyce
Moraceae
Anthelmintic
Western Amazonia
i
Flindersia maculosa
Rutaceae
Diarrhea
Australia
h
Gounia longipetala
Rhamnaceae
Antifungal, venereal diseases
Tropical West Africa
b
Grevillia heliosperma
Proteaceae
Skin infections
Australia
c
Hakea suberea
Proteaceae
Skin infections, antifungal
Australia
c
Hedycarya solomonensis
Monimiaceae
Skin infections
Solomon Islands
d
Heliciopsis artocarpoides
Proteaceae
Eye infections
Borneo
d
Laurelia novae-zealandiae
Monimiaceae
Gonorrhea
New Zealand
d
Litsea glutinosa
Lauraceae
Antifungal, skin infections
Northern Thailand
a
Table 1.1 (cont’d) Plant
Family
Ethnobotanical Use
Location
Ref.
Litsea glutinosa
Lauraceae
Skin infections
Australia
h
Macaranga denticulata
Euphorbiaceae
Food poisoning
Northern Thailand
a
Mallotus oppositifolia
Euphorbiaceae
Dysentery
Tropical West Africa
b
Melaleuca acacioides
Myrtaceae
Respiratory ailments
Australia
c
Melaleuca alternifolia
Myrtaceae
Skin infections, antifungal
Australia
h
Melicope monophylla
Rutaceae
Skin infections
Philippines
d
Mimosa albida
Mimosaceae
Respiratory ailments
Western Amazonia
g
Myrcianthes fragrans
Myrtaceae
Influenza
Bahamas
d
Myrciaria tenella
Myrtaceae
Hemorrhage
Eastern Amazonia
e
Neolitsea umbrosa
Lauraceae
Skin infections
India
d
Omalanthus populifolius
Euphorbiaceae
Hemorrhage
Australia
h
Piper novae-hollandiae
Piperaceae
Gonorrhea
Australia
h
Piper betle
Piperaceae
Skin infections
Northern Thailand
a
Table 1.1 (cont’d)
a
Plant
Family
Ethnobotanical Use
Location
Ref.
Piper capense
Piperaceae
Anthelmintic
Tropical West Africa
b
Piper ottonoides
Piperaceae
Toothache
Eastern Amazonia
e
Polyscias fruticosa
Araliaceae
Chest pains
Northern Thailand
a
Siparuna budleiaefolia
Monimiaceae
Arthritis
Western Amazonia
g
Sloanea amygdalina
Elaeocarpaceae
Analgesic
Haiti
d
Syncarpia hillii
Myrtaceae
Skin infections
Australia
h
Syzygium guineense
Myrtaceae
Tonic
Tropical West Africa
b
Syzygium suborbiculare
Myrtaceae
Stomach pain
Australia
h
Zanthoxyllum gilletii
Rutaceae
Antibacterial
Southeastern Africa
b
Zanthoxyllum juniperina
Rutaceae
Toothache
Eastern Amazonia
e
Zanthoxyllum schinifolium
Rutaceae
Anthelmintic
China
f
Zanthoxylum clava-herculis
Rutaceae
Gonorrhea
Native Americans
d
Anderson, 1993; b Iwu, 1993; c Barr et al., 1993; d Beckstrom-Sternberg and Duke, 1994; e Balée, 1994; f Huang, 1992; g Martinez, 1993; h Lassak and McCarthy, 1990; i Iglesias, 1993
1.2
Prospects for future medicines from natural sources
1.2.1 Antibiotic resistance Recently, established antibiotics have become less effective against many infectious organisms. In 1994 the Centers for Disease Control expressed concern over the possibility of a “post antibiotic era.” This concern is heightened by our tenuous ability to detect, contain, and prevent emerging diseases. The emergence of pathogenic microbes with increased resistance to existing antibiotics provides a major incentive for the discovery of new antimicrobial agents. The problems of drug-resistant pathogens have been reviewed recently (Anderson, 1999; Levin and Andreasen, 1999); there is pressing need for more effective antibacterial therapy. Based on several recent reports, pathogens of immediate concern are methicillin-resistant Staphylococcus aureus, a common cause of hospital infections, which is evolving a resistance to vancomycin (Rotun et al., 1999; Sieradzki et al., 1999); Pseudomonas aeruginosa in which multidrug resistance has become problematic (Hsueh et al., 1998); Streptococcus pneumoniae (Schwartz, 1999), Haemophilus influenzae (Dominguez and Pallares, 1998) and Moraxella catarrhalis (Budhani and Struthers, 1998; McGregor et al., 1998), common respiratory pathogens in which penicillin resistance is spreading; multidrug-resistant strains of Mycobacterium tuberculosis (Ginsberg, 1998), which are causing an alarming increase in the incidence of tuberculosis; enterococci, second only to Escherichia coli as a cause of nosocomial infections, which are developing resistance to β-lactams, aminoglycosides, and vancomycin (Low et al., 1995; Weber et al., 1999; Fleenor-Ford et al., 1999). In addition, enteric pathogens Shigella spp. and Salmonella spp. (Gaudreau and Turgeon, 1997) are now showing resistance to antibiotics, as are sexually transmitted
organisms, particularly Neisseria gonorrheae (Ehret and Judson, 1998). In this thesis project, screening for antibacterial activity has been carried out against the following representative organisms: Gram-positive bacteria, Bacillus cereus, Staphylococcus aureus, and Streptococcus pneumoniae; Gram-negative bacteria, Pseudomonas aeruginosa and Escherichia coli.
1.2.2 Cancer chemotherapy There are four major approaches to the treatment of cancer: surgery, radiotherapy, immunotherapy, and chemotherapy. The limitations of chemotherapy include the ability of cancer cells to develop resistance to anticancer drugs and the fact that many normal cells are destroyed in addition to the tumor cells. Because of these limitations, there is a need to find new antitumor drugs that can exploit the differences between normal and cancerous cells and selectively kill the cancer cells. Additionally, these new antineoplastic agents can be used in conjunction with existing anticancer protocols or in combination with other anticancer drugs. In the United States, lung cancer represents 14% of yearly cancers, breast cancers constitute 14% (30% of cancers in women), prostate cancer accounts for 17% (35% of cancers in men), colon/rectal cancers account for 10%, bladder cancer makes up 4%, skin cancers account for 3%, and liver cancers are 1% (Landis et al., 1998). However, hepatocellular carcinoma accounts for approximately 5-15% of worldwide cancer incidence, due to increased rates of infection by hepatitis B virus in other countries (Cooper, 1992). Natural products have the potential to afford new chemical structural types with novel mechanisms of activity; it is important to explore these resources. Plant
extracts in this project have been screened for in-vitro cytotoxic activity against the human tumor cell lines: Hep-G2 (hepatocellular carcinoma), MDA-MB-231 (mammary adenocarcinoma), Hs 578T (mammary ductal carcinoma), SK-Mel-28 (melanoma), A431 (epidermoid carcinoma), and 5637 (primary bladder carcinoma).
1.2.3 Emerging infectious diseases The threat of emerging infectious diseases, either widely presumed to be under control or viewed as new or “exotic” infections, is increasing. This may be attributed to overpopulation, expanding poverty, urban migration, and international travel. These newly emergent infectious diseases may result from mutation of existing organisms; known diseases may spread to new populations or geographical areas; new, previously unrecognized diseases may appear in humans due to transmission from insects, animals, or other environmental sources. Recent examples of emerging infectious diseases in the United States include West Nile viral encephalitis (Centers for Disease Control, 1999), hantavirus pulmonary syndrome (Engelthaler et al., 1999), coccidioidomycosis (Kirkland and Fierer, 1996), cryptosporidiosis (Colley, 1995), and Escherichia coli O157:H7 infection (Feng, 1995). Development of antibiotic resistance is one cause for the increase in emerging bacterial infection (Levy et al., 1997). Also, the incidence of opportunistic infection continues to increase rapidly because of the increased number of immunocompromised patients with immune deficit due to factors such as inherited disease, aging, premature infancy, HIV infection, radiation and cancer chemotherapy, immunosuppressive drugs during transplantation, malnutrition, pregnancy, severe trauma, burns, concurrent
infection, or malignancy (Warnock and Richardson, 1991). This has created a need for more effective treatment for otherwise relatively benign pathogens. Opportunistic fungal infections are a major cause of disease and death in immunocompromised patients (Connolly et al., 1999; Warnock and Richardson, 1991). Examples of organisms responsible for opportunistic mycoses include Candida albicans, Cryptococcus neoformans, Malassezia furfur, Aspergillus spp., and Mucor spp. Screening for antifungal activity against the pathogenic yeast Candida albicans has been included in this work. Viral diseases remain one of the largest contributors, worldwide, to human disability and death (Flint et al., 1999). Although immunization has proven to be useful in controlling a number of viral diseases, there are many viruses such as herpes viruses, hepatitis C virus, influenza viruses, and a disturbing number of emerging, or “exotic” pathogenic viruses such as human immunodeficiency viruses, dengue viruses, Hantaan hemorrhagic fever virus, and Ebola virus, which do not have effective vaccines (Holland, 1998). The specter of new and recurring diseases caused by viral pathogens such as these underscores the urgent need for new, diverse antiviral agents. Resistance to antiviral drugs is also becoming problematic. Antiviral agents for the treatment of herpes infections such as acyclovir, valacyclovir, and foscarnet have had a major impact on the management of these diseases (Baker, 1998), and acyclovir still remains the treatment of choice for primary and recurrent herpes simplex (HSV-1 and HSV-2) and herpes zoster (shingles) infections. However, acyclovir-resistant strains of HSV-1 and HSV-2 (Saijo et al., 1998; Pechere et al., 1998) illustrate the need for development of new antiviral agents against these viruses. Barrish and Zahler (1993)
have asserted that viral pathogens encode only a limited number of essential proteins and an even smaller number of essential enzymes that are amenable to “rational design” of low molecular weight inhibitors, so screening of natural products will continue to play an important role in the future of antiviral research. The need for novel classes of antiviral agents with novel mechanisms of viral inhibition is becoming increasingly important in order to combat new nucleoside-resistant mutants. This study reports screening of Australian rainforest plant extracts against Herpes simplex virus type 1 carried out by Professor Glenn Gentry at the University of Mississippi Medical Center.
1.3
Tropical rainforests as sources of medicines Tropical areas of the world are incredibly species rich. More than half of the
species of plants and animals worldwide are found in tropical rainforest regions. As an illustration of this species diversity, one can compare the 30 tree species per hectare found in the Appalachians, the most species rich region of the United States, to the average 40 to 100 tree species per hectare found in tropical rainforests (Kricher, 1997). The humid tropical region of North Queensland, Australia has about 6000 km2 of extant rainforest with 740 species of trees occurring in these tropical rainforests (Tracey, 1982). Only about 15% of known plant species have been examined for potential medicinal utility. Thus, 85% of known species as well as the huge number of undescribed species have yet to be investigated for new phytopharmaceutical compounds. The tremendous species diversity in the tropics leads to increased competition between species; in order to survive, individuals must compete for available space and resources. The same tropical environment which leads to such great plant diversity also
leads to enormous diversity in herbivores and phytopathogens (fungi, bacteria, nematodes, etc.). Thus, tropical plants have evolved systems of defense against these predators. A major means of defense available to plants is defensive chemicals (secondary metabolites) which can be exploited by the bioprospector searching for new medicines. Over millions of years, tropical plant species have performed complex combinatorial chemistry and biological testing in order to develop the best defensive chemicals at the lowest energy and resource cost to the plant itself. Australia has a unique and distinctive flora. Although Australian rainforests are limited in distribution, there is a very high proportion of endemic species and genera. In the late Jurassic period (about 160 million years ago), west Gondwana (Africa and South America) started to separate from east Gondwana (Australia, Antarctica, India, and Madagascar). At the final breakup of Gondwana (about 45 million years ago), the Australian continent split away from Antarctica. Australia was, therefore, effectively isolated from the rest of the world until it drifted northward and contacted the southeast Asia plate about 15 million years ago. Thus, Australia had 30 million years of evolutionary isolation. The Jurassic flora of Australia appears to have been very similar to that in other parts of the world (Adam, 1992), but latitudinal and climatic changes resulted in a high degree of ecological sifting. Although there was likely a post-Miocene invasion (and subsequent speciation) of plant taxa from the north, accounting for many taxonomic similarities between northern Australia and Malesia, many Australian rainforest taxa have a long history in Australia, and the isolation of Australia has apparently led to high generic endemism.
There is some information about the chemical and medicinal properties of Australian rainforest plants. Lassack and McCarthy (1990) have compiled a list of Australian medicinal plants, especially those used in Aboriginal traditional medicine. The Commonwealth Scientific and Industrial Research Organisation (C.S.I.R.O.) carried out an alkaloid and cytotoxicity screening survey of a number of Australian plants (including temperate as well as tropical taxa) from the mid-1940’s to the mid-1980’s (Collins et al., 1990). The work described in this thesis has focused on tropical rainforest plants from the Paluma rainforest that are taxonomically and evolutionarily unique, but that have been overlooked in terms of screening for biomedicinal applications.
