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CHAPTER I
INTROD UCTION
1 .I PLANTS AND HEALTH CARE
Plants have been used from the early civilization onwards as a source
of medicine for all types of diseases. In spite of recent development in the
synthetic drug chemistry and production of antibiotics, plants still occupy an
important role in the modern and traditional systems in all over the world.
Modern medicines are primarily from synthetic or plant origin and they may
have toxic effects while the plant medicines have less toxicity and their
importance being realized in both developed and developing countries. It is
expected that in future the plant products will play a major role in the
healthcare programme of all countries.
India is a subcontinent with several medicinal plants. Ancient Indians
used plants in the Ayurvedic system of medicines. An intensive study of
indigenous drug plants and their therapeutic potential may be given useful
remedies and widened the scope of traditional systems of Medicine. The
drug Vincristine and Vinblastine was isolated in 1961 from Vinca rosea plant
and this is now being used for the treatment of cancer throughout the world.
The Central drug research laboratories in collaboration with National
Institute of Health U.S.A screened several plants. Many of the plants were
screened and there is at present a long list of plants claim to have anti
cancer properties (Hartwell-1968-71). A few compounds like taxol.
hornoharringtonine and ellipticine have been progressed to the point of
clinical trial and are used as medicines. Other examples are bteomycin,
comptothecin, bryostatin and Phorbolesters are used (Suffness and Pezzuto
1991). Table-1 contains Indian, Chinese and South American plants used
against cancer.
1.2 Cancer
According to the definition of the International Union against Cancer
'Cancer is a disturbance of growth characterized primarily by an excessive
proliferation of cells without apparent relation to the physiological demands
of the organ involved'. Cancer is still a dreaded disease, which accounts for
9% of the deaths throughout the world. It is one of the 10 leading causes of
death today, in India. Cancer can occur in any organ of the body and the
cancer cell will tend to resemble their normal tissue of origin. Cancers of
various organs are quite different from one another and even within organs;
there are many different subtypes. It is, therefore, necessary to discover
separate drugs for the various types of cancer (Suffness and Pezzuto 1991).
Table-1
Anticancer plants used in the Indian, the Chinese and the South
American systems of medicine
Indian* Chinese*' S u t h American*** Acronychia barberi Actinida chinensis Aliiurn cepa
Ailanthus malabarica Agrimonia pilosa Cannabis sativa
Cissampelos pareira Belancanda chinensis Chenopodium
Mappia foetida Brucea ja vanica An thelmin ticum
Maytenus serra ta Curcuma aromatics Coffea <ara bi ca
Plumbago zeylanica Dysosma pleian tha Hyoscyamus albus
Podophyllum emodi Fagopyron dibo ttys lpomoea ba tatasa.
Podophyllurn pelta turn Hedyo tis diffusa Musa pa radisiaca
Sola n urn dulca ma rra Houttuynia cordata Ocim urn ba silicum
Solanurn trilobatum L onicera japonica Papaver somniferum
Sola n urn tripattiurn Podop hyllum emodi Pe troselin urn crisp urn
Tylophora asthma tica Ra bdonia rubescens Rosemarinus officinalis
Xanthiurn strumarium Sophora flavescens Vinca rosea
'Venugopalan 1987 ""Sun and Xiao 1994 ***Duke 1986
1.2.1 Cancer Treatment Modalities
Cancer is at present treated by various techniques like surgery,
radiotherapy, chemotherapy or a combination of all. Surgical techniques are
useful only in the area of primary tumour and not the neoplasm outside the
operation field. kadiation therapy from radiation sources of Cobalt, Cesium
or Iodine is used in the management of cancer. The complex field of
chemotherapy is new and the growth is very rapid. The commonly used
therapeutic agents are anti tumour antibiotics, alkylating agents, nitrosoures,
anti metabo[ites, hormones and natural products. Phospholipid vesicles like
liposomes carry out drug delivery at the site of tumour. Magnetic albumin
micro spheres and monoclonal antibodies (Haskel 1 985). lmmunotherapy
can increase the general imrnunocornpetence of both cell mediated and
hormonal immunity like lymphokine, interferon's and interleukins. Biological
response modifiers are agents that alter the immune response of host
defense mechanism. Tumour means a swell or lump in the body resulting in
the growth of tissue or collection of fluid. This may be benign or malignant. A
neoplasm is abnormal growth in the body, which may be benign or
malignant.
1. Clonality: In most cases, cancer originates from a single stem cell,
which proliferates to farm a clone of malignant cells.
2. Autonomy: Growth is not properly regulated by the normal
biochemical and physical influences in the environment.
3. Anaplasia: There is a lack of normal coordinated cell differentiation.
4. Metastasis: Cancer cells develop the capacity for discontinuous
growth and dissemination to other parts of the body.
The process by which a normal cell is converted into one which
exhibits these characteristic traits is termed malignant transformation.
There are over 150 recognized types of cancer in man. The two main
types are carcinomas and sarcomas. 'Carcinomas' refer to cancers of the
epithelial tissue, that cover the external surfaces of the body including
cancers of skin and breast and the internal surfaces including the alimentary
tract and those organs derived from the embryo's gut. The latter includes
cancers of mucous membrane, liver, pancreas, intestine, prostrate and
thyroid. 'Sarcoma' is a term that applies to cancers of connective tissue such
as muscle, cartilage, bone and fibrous binding tissue. Leukaemic lymphomas
and other cancers of the blood forming cells' are classified separately
because they are systematic diseases. Gliomas are tumours of nerve tissue,
melanomas, cancers of the skin. It is the tissues of origin, not the organ of
origin on which the properties of the tumour depend. Each type of cancer
has a relatively distinctive natural history that describes the likely course of
the particular neoplastic tumour process.
Since all cells in an organism originate from a single fertilized egg
(zygote), all carry identical genetic information. The proliferation and
differentiation of this cell into an embryo and eventually into an organism
involves selective and coordinated expression of the genomic machinery.
This includes information, which permits cells to expand clonally, to function
with various degrees of autonomy, to differentiate and dedifferentiate and to
move from one part of the organism to another in a coordinated way. In the
case of malignancy, the normal control process is bye-passes (Mendelson
1991). Cancers are very close in properties to their tissues of origin, and as
such have few differences to be exploited in drug design.
1.3 Development of a drug for cancer
The targets for chemotherapy that are currently recognized include
anything having to do with cell growth and cell division i.e., replication in all
its details-transcription, translation, mitosis, the cell cycle metastasis, the
process by which cells break off from the parent malignant tumour and travel
to distant sites through the circulation where they invade and set up new foci
of tumours (Suffness and Pezzuto 1991).
It is possible to search for new drugs that interact with useful targets
that are currently undefined since it really is not necessary to know the target
if the effect of hitting the target can be predicted. Alternatively, a screen can
be designed to monitor a desired response even if the target responsible for
mediating that process is not known. The major problem with cell growth and
division as the target is that in any organism, there are growing and dividing
normal cells which will be harmed by drugs acting on this basis.
Furthermore, there are some tissues whose cells have rapid turnover rate
and these tissues will be extremely sensitive to agents, which affect dividing
cells. Most prominent of these are bone marrow and gastrointestinal
epithelium, which are the most frequent targets of drugs used in cancer
chemotherapy that yield toxicological response. An ideal drug is one, which
is so highly selective for tumour tissue that it can kill or incapacitate tumour
cells while not affecting normal tissue. The dose of chemotherapeutic drug
administered should be such that the rapidly dividing tumour cells incur
maximum damage while normal tissue remains unharmed to enable the
system to recover.
Cell function and differentiation are the outcome of multiple and
complex events (Stockdale and Topper 1966). Genes receive information,
which modulate their activity. Many different molecules are able to bind to
nucleic acids (DNA, RNA), there by modifying gene activity and that of
various enzymes connected with it. It is well established that the effect of
endogenous or exogenous molecules on DNA replication may dramatically
affect other biochemical processes, thus influencing cell life. The aim of
cancer therapy should be for selecting molecules capable of specifically
correcting or arresting cell disturbances at the nucleic acid level and
enhancing beneficial gene expression. Thus, the challenge of the cancer cell
must be met on two different levels (1). Cancer cell multiplication must be
selectively arrested without injury to normal cell (2). Competence of the
immune system must be protected and restored for an active defense of the
body.
Beljanski's group tested several specific anticancer compounds,
mostly plant derived alkaloids, flavanones and flavonoids, which selectively
restabilise cancer DNA, yet have no effect on normal DNA. They do not
affect normal DNA replication but they bind no replication-initiation sites or
prevent chain elongation of cancer DNA. They incapacitate highly malignant
cells, but can revert to normal, cells in which malignant transformation has
not gone too far. They have proved active in both mammalian and plant cells
(Beljanski M and Beljanski MG1982, Beljanski et a1 1982, Beljanski Mi993).
The present trend in the development of cancer medicine leads to
isolation of active principles in the form of chemicals, which may be active or
also may be toxic. In the Ayurvedic system of medicine, several plant
products mixed together and given as medicines. The principle behind this is
the toxic effect of one of the plant is nullified by the presence of other plant
products, which if given separately may be toxic. Hence, toxicity is avoided
in the system of medicine. However, pure compounds isolated may be free
from toxicity or toxic and based on this structure, synthetic compounds may
be developed which may proved to be more potential in treating cancer.
Work in these lines is also in progress.
1.3.1 Development of anticancer drugs from plants
There are several medicines at present available for the treatment of
cancer and 25 to 30% are from plant origin. These cornp~und~s have a wide
variety of novel structure and mechanism of action. (Cassady 1 990).
According to Hartwell 1971, it has been estimated that more than 3000
species of plants have been used throughout the world to treat cancer. The
significance of folklore medicines is relevant to the present day since we
have got an anti tumour compound podophyllotoxin from the root of the plant
podophyllum peltatum based on the information obtained from folklore
medicines. The novel terpeniod taxol has become an important anticancer
compound and this is obtained from the screening of natural products
(Kingston 1990).
The other compounds used are drugs like mytomycin, anthramycin
and other natural products even these drugs have limited scope with side
effects. The search for anticancer compounds from natural products still
continues. Some of the important anti cancer compounds are given in Table-
2. This thesis consists of investigations on anticancer property of
B.racemosa Raxb seed extract based on information from folklore
medicines. The following stages are to be followed in the development of
anticancer drug from plant.
1. Collection and identification of plants based on available information
of use against tumour.
2. Preparation of plant extracts
3. Primary screening of plants for antiturnour activity.
4. Large scale collection of active plants
5. Bioassay directed fractionation to isolate pure active compounds
6. Structural elucidation of the active compound(s)
7. Tumour panel testing
8. Large scale production for drug development
9. Preclinical study (Formulation, Pharmacology, Toxicology)
I0,ClinicaI trials
Some of the most important antineoplastic agents produced by plants
(Misawa et a1 1985, Cosmo and Miswal995)
1. Baccharin I
2. Bruceantin
3. Camptothecin
4. Cesalin
5. 3-Deoxycolchicine
6. Podophyllotoxin
7. Ellipticin
8. Fagaronine
9. Harringtonine, Homoharringtonine
10. Indicine-n-oxide
11. Maytansine
12.TaxoI
13. Thalicarpine
14. Triptolide, Tripdiolide
15, Vincristine, Vinblastine
Antineoplastic agents
Baccharis megapotarnica
Brucea an tidys en terica
Camptotheca acuminata
Ca esa lpinia gilliesii
Colchicum speciosum
Podophyllum pelta tum
Ochrosia moorei
Plants
Fagara zanthoxyloides
Cephalotaxus harringtonia
He lio tropium indcum
Put te rlickia ve rrucosa
Taxus b re vifolia
Thalictrum dasycarpum
Trip te rygium wildfo rdii
Vinca rosea
1.3.2 Bioassays of plant products
Any information about anticancer activity of plants can be obtained
only by using suitable assay procedure on the plant products. This can be
carried out by several methods using cancer cell-lines; the effect of this drug
on the growth of this cell-line rather the preventive effect of the growth can
be determined. Without an effective assay system, it is not possible to
evaluate the effect of the drug either for treatment in experimental animals or
estimating at various stages of the purification process (Cassady et al1988).
Each step of fractionation and isolation of bioactive compounds from plant
extract is guided by both in vitro technique using human epidermoid
carcinoma (KB) or murine lymphocytic leukemia cells in culture and in vivo
techniques using mice bearing transplantable leukemic (p-388) and solid
tumour derived from murine as well as human sources.
Recently, the NCI, USA has developed new anticancer drugs
screening programme based on the use of multiple panels of well
characterized human tumour cell lines representing the key human tumour
types. This is to discover agents with high selectivity for the major forms of
human solid tumours (lung, colon, ovarian, breast, renal, melanoma and
central nervous system). Agents showing differential or selective activity of
in vitro growth inhibition will be subsequently evaluated by in vivo tests in
mice bearing the same human tumour cell lines found sensitive in vitro. This
new in vitro-in vivo screening is closer to the real human tumour situation
than those used previously and therefore the drugs discovered by this
screening may be expected to be more predictive of clinical activity than
drugs discovered by the older screening methods.
The Central Drug Research Institute at Lucknow has carried out
investigations of plants for bioactivity including anticancer property in
collaboration with NCI, USA.
1.4. Plants used in the treatment of Cancer '
a
The classic anticancer drugs do not distinguish normal cells from
cancer cells. Using a sensitive biochemical test (oncotest), some plant
alkaloids and flavanones capable of distinguishing in vivo and in vitro human
and animal cancer cells from normal ones were isolated. These prevent in
vitro proliferative capacity of cancer cells only. They bind to cancer DNA and
ignore normal DNA. They inhibit tumour development in mice (Beljanski and
Beljanski, 1 982). They exhibit a strong synergistic effect with classic
anticancer compounds such as cyclophosphamide (Beljanski 1993).
In recent years, several plants have been reported to be efficient
cytotoxic and antitumour agents. Inhibitors of chemical carcinogenesis can
also have a profound effect on the expression of experimental tumours
(Wattenberg 1985). Such inhibitors are seen in the plant kingdom. Most of
the present day, drugs used in chemotherapy are toxic to normal cells,
leading to unwanted side effects. Therefore, it is important to search for new
compounds, which can reduce the harmful effects of anticancer drugs to
normal tissues. lmrnunostimulation is a therapeutic concept, which aims at
the stimulation of our nonspecific immune system. In other words, it is the
nonantigen-dependent stimulation of function and efficiency of granulocytes,
macrophages, complement and natural killer cells in contrast to immunity
achieved by immunization. This type of unspecific irnrnunostimulation
constitutes an alternative or adjuvant for conventional chemotherapy of
tumours. In the light of these factors, the plans used for cancer are dealt with
under the following subdivisions.
1.4.1 Cytotoxic and antitumour principles
A whole gamut of phytochemicals has been reported with cytotoxic
and antitumour activities. Mushrooms have been advocated for treatment of
cancer (Mascarenhas 1994). Although some medicinal uses are known for
ferns, a art well's extensive analysis (Hartwell 1968, 1970, 1971) and studies
7 lao'% 11
~esearch Institute, Lucknow, India
(Rastogi and Dhawan1982) suggest possible chemotherapeutic activity for
several ferns.
The biochemical effects of these naturally occurring inhibitors of
carcinogen sis may result from various biochemical alternations in the target
organs. Other potential chemo preventive agents include reserpine,
curcumin, glabrene, and certain fatty acids (Cassady 1 990). Various
flavonoids have previously been shown to have anticarcinogenic activity.
Watten berg and co-workers (1 985) demonstrated that the flavonoids rutin,
morin and quercetin inhibited tumourigenesis by B (a) P in mouse skin. Two
common flavonoids, apigenin and luteolin are active principles of antiturnour
action in vitro against five, human tumour cell lines (Ryu et al 1994).
(Elangovan et a/ 1994) studied the in vitro effects of bioflavonoids on human
larynx carcinoma (HEP-2) and S-180 cell lines. Depletion of glutathione in
the cells and loss of cell viability was observed in the quercetin treated
cultures. Depletion of glutathione leads to increased accumulation and loss
of cell viability (Axe lson and Mannervik 1983). Proanthocyanidins like those
seen in Cistus incanus are polyflavonoids possessing anticarcinogenic
activity. Dimeric and trimeric procyanidins, the most common type of
proanthocyanidins, mimic inhibition of biochemical markers of TPA (12-0-
tetradecanoyl-phorbol-13-acetate) induced tumour promotion.
Alpha-hederin, a triterpenoid saponin induced vacuolisation of
cytoplasm and membrane alterations leading to cell death and caused
impairment of phospholipid metabolism (Danloy et a1 1994). Flavonoids have
been reported to serve as scavengers of reactive species of oxygen such as
superoxide singlet oxygen and H202 (Takahama 1985) and are therefore
considered free radical scavengers (Princemall and Goutier 1985). Over 500
varieties of flavonoids are known (Havsteeri 1983). Other effects of
flavonoids on cell function include antimutagenic (Alldrick et a1 1986) and
cytotoxic (Hirano 1989). One mechanism by which plant flavonoids can
inhibit ultimate carcinogenic metabolites is direct reaction with 'these
metabolites resulting in their detoxification.
In Gymnosporia rothiana, the active principle was shown to inhibit the
biosynthesis of macromolecules, which subsequently led to inhibition of
RNA, and DNA biosynthesis (Chapekar and Sahasrabudhe 1981). In Crocus
sativus, thymidine uptake studies indicated the mechanism of action to be
inhibition of DNA synthesis (Nair et al 1991 b) and modulation of functional
levels of other antioxidants (Nair et a1 1994).
Some hydrophobic constituents like steroids in the stinging nettle
roots (Urtica dioica) in hi bited membrane Na+ K' ATPase activity, which later
suppressed prostrate cell metabolism and growth (Hirano eta'/ 1994).
Ginsenosides were able to increase the bioactivity of recombinant
tumour necrosis factor in vivo and in vitro (Chun et a1 1991). Lu and Kunreu
(1 987) suggested that anticancer drugs might increase intracellular cyclic
AMP levels by in hi biting 3'5' c-AMP-PDE (NTK') ATPase activity to
modulate the proliferation and differentiation of cancer cells.
Jawaharene, an antiturnour principle from Aspergillus hampered the
metabolism and normal growth rate of treated mice, though significant
effects were not seen on haematology of mice (Das and Ray 1988). Partially
purified bromelain is used as an adjunct in chemotherapy. It is suggested
that bromelain may lyse blocking tumour antigen antibody complexes which
otherwise would prevent attack of cytotoxic T-lymphocytes. Bromelain may
also unmask blocked tumour surface antigens, leading to complement
binding and cytolysis (Maurer et a1 1988). Bromelain is able in vitro to induce
leukaemic cells to differentiate into mature granulocytes, macrophages and
to reduce tumour growth. These properties of bromelain should warrant
application of bromelain as an adjunct in combination therapy of tumours.
Bioassay directed fractionation of leaf oil of Petroselinum sativum
yielded myristicin. It has ability to induce activity of detoxifying enzyme
systems (Zheng et a1 1992).
The methanolic extract of clove, Syzygium aromaticum, showed
remarkable induction of differentiation of mouse myeloid leukaemic cell line
(MI) into macrophage like cells. Oleanolic acid and crategolic acid were
isolated from the extract (Umehari et a1 1992). The rhizome of Curcumin, the
major constituent of turmeric was proved to interact with ca2'-calmodulin
complex, and thus modulate biological activities like inhibition of tumour
promotion (Nishio et a1 1992).
Antitumour property of Parthenium hysterophorus was due to its
capacity to modulate biotransforming enzymes in transplanted murine
leukaemias. The glutathione levels and cytochrome P-450 contents were
altered, leading to slow development of tumours (Mukherjee and Chatterjee
1 993). Indiru bicin, the component responsible for anticancer activity of
lndigofera tinctoria, yielded marked inhibition on Lewis lung carcinoma and
Walker carcinoma (Xiujuan et a1 1981).
Table 3 gives the most important cytotoxic and antiturnour plants
studied and the bioactive principles, involved in the antiturnour activity.
Table - 3
Cytotoxic and antitumour agents isolated from plants
Reference
Krishnaswam
y et a1 (1 991)
Tanessig et
a1 (1 985)
Mizuno et al
(1 994)
c.f: Cassady
(1 990)
Wondenberg
et a1 (1 994)
Zheng (1994)
Das and Ray
(1 988)
Ghosh et a1
(1 994)
Action
tumour regression
cytatoxic to Lewis lung
carcinomaYC-8
lymphoma; MCA-1
ascitic tumour cells
cytotoxicto HeLa cells
cytotoxic to leukaemic
cells and HT-29 colon
tumour cells
cytotoxic to COLO -
320 and human lung
carcinoma cell-line
cytotoxic to P-388 A-
549, HT-29,MCF and
KB
antiturnour
antitumour to DLA
Plant
Alstonia scholaris
Ananas comosus
Angelica edulis
Annaona
densicoma
Arnica Montana
A.charnissonis
Altemisia annua
A spe rgillus sp
Beta vulgaris
Bioactive Principles
alakaloid (echitarnin
chloride)
bromelain
coumarins
polyketides
Sesquitepene
lactones and
f lavonoids
terpenoid,
artimisinin and
f lavonoids
jawaharene
B-carotenes
Bolbostemrna
panicaturn
Brucea Ja vanica
Camellia thea
Limonium axillare
Cryptolepis
sanguinolenta
Crocus sativus
Lindera
megap hylla
Peganum harmala
Annona purpurea
tu beimosides
quassinoid
glaycosides
flavonols
new flavonol
glycoside
cryptolepine 8
neocryptolepine
gl ycoconjugate
saponin
alkaloid
purpuracenin
antitumour
antitumour to EAC
Walker-256 carcinoma,
P-388
antitumour to EAC
inhibition on EAC cells
cytotoxic to tumour
cells and
topoisomerase I I
in hibitor
cytotoxic to tumour
cells
inhibition of HUH - 7
(hepatoma cell line)
cytotoxic activity
anti tumour activity
potent cytotoxic activity
against human solid
tumour cell lines
Sharma and
Agarwal
(1 992)
Sur and
Ganguly
(1 994)
Kandil - FE
el a1 (2000)
Bailly -G et
a1 (2000)
Escribano - J
et a1 (2000)
Huang - RL
et a1 (1 998)
lkeda - T et
a1 (1 997)
Lamchouri -
F et a1 (1 999)
Chavez - D
et a1 (1 999)
1.5 Apoptosis
Apoptosis or programmed cell death is characterized by certain
distinct morphological and biochemical features. Most chemotherapeutic
drugs exert their antitumour effects by inducing apoptosis. Therefore, an
effective compound inducing apoptosis appears to be a relevant strategy to
suppress various human tumours. The morphological process of cells
Leontodon
hispic us
Kalopanax pictus
Poly lepis
racemosa
Ekmanianthe
longiflora
Ajuga decumbens
Polypodium
leucotornos
Sedum
sarmentosum
cytotoxic activity
against solid turnour
cell line
anti mutagenic &
cytotoxic activity
cytotoxic against M-14
melanoma and ME 180
cewical carcinoma
cytotoxicity against
human cancer cell lines
cherno preventive
activity
inhibit production of
cytokines
anti proliferative effect
sesquiterpene
lactone
hederagenin
m~nodesmosides
bisdesmo sides
ursolic acid, pomolic
acid
2-acetylnaphthol
2,3-bifuran-4-one
cyasterone and 8-
acetyl harpagide
extract of ferm
alkaloid
Zidorn - C et
a1 (1 999)
Lee - KT et
a1 (2000)
Neto - CC et
a1 (2000)
Peraza - Sanchez - SR et a1
(2000)
Takasaki - M et a1 (1 999)
Gonzalez - S
et a1 (2000)
Kang- TH et
al(2000)
undergoing programmed cell death is called apoptosis and is characterized
morphologically by cytoplasmic shrinkage, active membrane blebbing,
chromatin condensation and fragmentation into membrane enclosed
vesicles. (Kerr JFR et a1 1972; Wyllie AH et a/ 1980). This visible
transformation is accompanied by biochemical changes. Those at the cell
surface include the externalisaton of phosphatidylserine and other
alternating that promote recognition by phagocytes. lntracellular changes
include the degradation of the chromosomal DNA into high molecular weight
and oligonucleosomal fragments, and cleavage of a specific subset of
cellular polypeptide (Kerr JFR et a1 1972; Wyllie AH et a1 1980). This
cleavage is accompanied by a family of intracellular proteases, called
caspases. Caspase activation is the biochemical event that, more than any
other, defines a cellular response as apoptosis.
During post embryonic development of the nematode Caenorhabditis
elegans, I31 of 1090 cells die in every organism. The isolation and molecular
characterization of the most important genes involved in this process, ced-3,
ced-4 and ced9 have shown a high degree of conservation of these killer
genes throughout the animal kingdom (Hotvitz HR et ai 1999). The protein
ced-3 is highly homologous to human caspases, and ced-9 is a functional
homologue of the human antiapoptotic protein. BCL2, APAFI (apoptotic
protease-activating factor 1) is a human horndlogue of ced-4 protein (Zou H
et a1 1997). Collectively, these findings underscore the high degree of
conservation of the cell death pathway from nematodes to human beings,
and suggest that a core death machinery exists in all cells (Fadeel B et a1
1999).
1.5.1 BCL2 family: Killers and protectors
The human BCL2 homologues make up the major apoptosis-
regulatory gene family. The apoptosis-suppressing BCL2 gene was
discovered as a proto-oncogene found at the breakpoints of t (14: 18)
chromosomal translocations in low grade B-ell non-Hodgkins lymphoma.
Antiapoptotic BCL2 like proteins can heterodimerise with proapoptotic
proteins of the BCL2 family, thus antagonizing them. An important link
between mitochondria1 and cytosolic apoptotic events was found when APAF
1 is a cytosolic protein that rests in a latent state until bound to cytochrome
C. This protein is commonly released from the mitochondria during apoptosis
induced by many stimuli (Green D et a1 1998). The resulting complex
associates with procaspases 9, resulting in a cascade of caspase activation
and apoptosis. Mitochondria play an important part in apoptosis. Several
apoptotic stimuli induce translocation of BAX from cytosol to mitochondria,
where it induces these organelles to release the caspase activating protein
cytochrome C. BAX seems to create pores in the outer membranes of
mitochondria of sufficient to allow cytochrome C to escape (Reed JC 1999).
The BCL2 family has also been implicates in resistance to therapy.
Anticancer drugs and radiation ultimately kill cancer cells by inducing
apoptosis. There is abundant evidence that BCL2 is a multidrug resistance
protein that prevents induction of apoptosis by radiation 'and almost all
chemotherapeutic agents in current clinical use (Reed JC 1995 la).
Conversely, decreased expression of BCL2 achieved by antisense methods
increases the susceptibility of cancer cells to apoptosis induction by many
chemotherapeutic drugs. Clinical trials are in progress to attempt
chemosensitisation of tumours with BCL2 antisense oligonucleotides (Koller
E et a1 2000). BCL2 genes are regulated by p53. The p53 protein has been
frequently implicated in response, to genotoxic stress injury, inducing cell-
cycle arrest and apoptosis when DNA is damaged by anticancer drugs or
radiation (Reed JC 1999 1 b).
1.5.1 Therapeutic implications
- The processes controlling apoptosis must be tightly regulated.
Consequently, the malfunction of the death machinery intrinsic to every cell
may have a primary or secondary role in various diseases, with essentially
too little or too much apoptosis leading to proliferative or degenerative
diseases. Apoptosis regulating therapies, which could be used alone or in
conjunction with conventional treatments, could include injectable molecules
targeted at upstream modulators of apoptosis, such as death receptors or
soluble death ligands, as well as small molecule pharmaceutical agents
designed to apoptosis-related genes and gene products. Inhibition of the
transcription, factor NF-kB through adenoviral delivery of I-kB sensitizes
chemoresistant tumours to treatment, resulting in regression (Wang WT et a1
1999). The inhibition of NF-kB and its associated antiapoptotic activity, which
may depend on the transcriptional activation of the survivin-related .
molecules CIAPI and CIAP2, could be useful as an adjuvant therapy in
cancer treatment (Fadeel B et a1 1999). Table-4 gives recent reports of the
injection of apoptosis by plants.
Table- 4
Apoptosis induced by plants
Reference
Lee - .IH et a/
(2000) -
KO-WG et a/
(2000)
Yoon - Y et al
(1 999)
Ren - W et al
(1 999)
Cui - B et a1
(1 999)
Plant
Rubus crataegi
folius
Vitexrotundfolia
Lithospermurn
e ryth ro rh izo ri
Solanum
murjca turn
Ra tibida
columinifera
Bio-active principle
methanol extract
polymethoxy
f lavonoids
shikonin
aqueous extract
xanthnolide
derivative
Action
induce apoptosis
and DNA
topoisomerase
ihi bitor
induce apoptosis
induce apoptosis
induce apoptosis
induce apoptosis
Ganoderma
tsuage
Alpiniaoxyphylla
miquel
Eriobotrya
ja ponica
Casearia arborea
Eugenia jambos L
Selagin e lla
delica tula
Eucalyptus
grandis
Adenophorea
trip h ylla
Ra tibida
columnifera
Croton cajucara
Physena
madagascariensis
Gan-KH et a1
(1 998)
Lee -E et a1 (1 998)
lto - R et a1
(2000)
Beutles et a1
(2000)
Yang-LL et a1
(2000)
Lin-L C et a1
(2000)
Takasaki-M et
a1 (2000)
Lee- I S eta1
(2000)
Cui - B et a1
( 1 999)
Grynberg et a1
(1 999)
Deng - Y et a1
(1 999)
lanostanoids and
steroids
extract
polyp henols
diterpene
hyd rolysable
tannins
bif lavonoids
euglobal - GI
ethyl acetate
fraction
sesquiterpenoids
19-nor-clerodane
diterpenes
triterpenes
induce apoptosis
induce apoptosis in
HL - 60 cells
cytotoxicity against
human oral turnout
cell lines
cytotoxicity
induction of
apoptosis
cytotoxity against
tumour cell lines
chemopreventive
action
induction of
apoptosis
induce apoptosis
anti tumour activity
cytotoxic against two
human breast
cancer cell lines
1.6 Chromosomes
Polygonium
cuspida turn
Aspidosperma
millia nsij
Uncaria
tomenfosa
Acanthupanax
senticosus
Some of the anticancer drugs induce chromosomal aberrations in
normal cells. A chromosomal change can occur due to various reasons such
resve rat ro I
ellipticine
indole alkaloids
sesamuri
as administration of drugs or the effect of some other factors. This induction
of chromosomal aberrations can be protected by a combination treatment
with plant-derived compounds. Bleomycin induced chromosomal breaks (CB)
induce apoptosis
induces
chromosome breaks
induce apoptosis
and inhibited
proliferation of
turnour cells
induce apoptosis
and suppress
growth
and sister chromatid exchange (SCE) in peripheral blood lymphocytes have
Kimura - Y et a1
(2001 )
Sakamoto-
Hojo-ET et a1
(1 998)
Sheng - Y et a1
(2000)
Hihasami - H
et a1 (2000)
been shown to be sensitive cytological markers for susceptibility to DNA
damage in patients with various types of cancer and in healthy controls
(Cloos J et a1 1999). Factors such as age, sex, smoking and alcohol
consumption could affect the values of some of these biomarkers and should
be considered as covariates when analyzing cytogenetic biomarkers
because these factors can affect the frequency of CB and SCE. The
genotoxic activity of taxol diluted in DMSO (Taxol-D) was studied by using
the micro screen prophase induction assay and sister chromatid exchange
analysis (SCE). Ethyl methane sulfonate (EMS) was used as a positive
control. Taxol D treatment did not induce significantly increased levels of
plaque forming units compared to the DMSO control (Mc Ghee EM and
Shankel DM 1993).
Chromosomal analysis requires examining cells in the process of
mitosis in which the chromosomal structure is most clearly defined.
Spontaneously dividing tissues such as occur in haematopoicess or in
cancer would be expected to provide mitotic cells for analysis even in a direct
harvest of the material submitted. Chromosomes are prepared on glass
slides and are treated by digestion (e.g. with trypsin) and then stained to
produce a banding pattern. The most commonly used staining techniques
are G-banding and K-banding, which produce characteristic staining or
banding pattern for each human chromosome. These techniques, in
combination with the physical chromosomal structure allow for the
identification of individual chromosome.
1.7.1 Modulators of the toxicity of the anticancer drugs vincristine and
cyclophosphamide
Considerable interest has been focused on compounds that might be
given along with antitumour drugs to reduce their dose limiting toxicity. For
these compounds to be useful, they must either be selectively absorbed by
nontumour cells or administered at an appropriate time before or after the
antitumour drug, when injury to tumour cells is irreversible and reversible to
nontumour cells e.g. MPG (mercaptopropionyl glycine) and WR 2721
(aminopropyl aminoethyl phosphorothioc acid (Milas et a1 1 984).
The treatments of many diseases owe much to the important
medicines that have been derived from plants and the treatment of cancer is
no exception. Unique classes of natural product anticancer drugs have been
derived from plants. As distinct from those agents derived from bacterial and
fungal sources, the plant product represented by the Vinca and Colchicum
alkaloids, as well as other plant derived products such as paclitaxol (Taxol)
and podophyllotoxin do not target DNA. Rather, they either interact with
intact microtubules, integral components of the cytoskeleton of the cell or
with their subunit molecules the tubulines. The vinca alkaloids are relatively
hydrophobic molecules that partition in to lipid bilayers in the uncharged
state, altering the structure and function of membranes. (Kremmer.T et a1
1980; Owellen RJ. et a1 1977; Ter-Minassian-Saraga et a1 1983; Ter-
Minassian-Saraga et a1 1981). Of their diverse effects, their only well-
documented direct action is disruption of microtubules, which results from
their reversible binding to tubulin, the subunit protein of microtubules. At
pharmacologically active concentrations, most of the biochemical effects
associated with exposure to the Vinca alkaloids are probably secondary to
disruption of microtubules although it is possible that drug induced changes
in lipid bilayers may alter some membrane dependent process. At high
intracellular concentrations, these compounds induce formation of large
crystalline aggregates that are composed of tubulin and drug. (Bensch K.G
et a1 1968; Bensch K.G 1969; Bryan J 1972) Despite their many biochemical
actions, the antineoplastic activity of the vinca alkaloids is usually attributed
to their ability to disrupt microtubules, causing dissolution of mitotic spindles
and metaphase arrest in dividing cells (Malawista SE. et al 1968 Ter-
Minnassian-Saraga L et a/ 1983 Bruchovsky N et a1 1965; George P et a1
1965; Howard SMH et a1 1980; Krishan A 1968; Lengsfeld AM et a1 1980;
Palmer CG et al 1960;). However, disruption of microtubules also leads to
toxicity in non mitotic neoplastic cells, and although the vinca alkaloids are
classified as mitotic inhibitors, their anti neoplastic activity in the clinical
treatment of cancer probably arises from perturbation of a variety of
microtubule-dependant process (Tucker RW et a/ 1977; Krishan A et al
1975; Madoc-Jones H et a1 1968; Madoc-Jones H et a1 1974; Shrek R 1974)
as well as from disruption of the cell cycle and induction of programmed cell
death (Shrek R et a1 1981; Tsukidate K et a1 1993) and exposure to vinca
alkaloids gives rise to diverse biologic effects, many of which could impair
essential functions, both in dividing and in non dividing cells (Harmon BV et
a1 1992;). Morphologic changes and cell death after treatment with VCR or
VLB have been seen in no dividing normal and leukemic lymphocytes, in
cultural leukemic cells during interphase, and in G and S-phase cells (Tucker
RW et a1 1977; Krishan A et a1 1975; Madoc-Jones H et a1 1968; Madoc-
Jones H et a1 1974; Schrek R 1974;). The vinca alkaloids also inhibit
secretary processes, apparently because of perturbations in membrane
trafficking with disruption of the cytoskeleton (Green LS et a1 1977;).
Platelets, which depend on the integrity of the peripheral ring of microtubules
for their discoidal shape, become spherical after treatment with Vince
alkaloids (Beck WT et a1 1986; Watkins RM et al1993;).
Cyclophosphamide remains an important antiturnour agent effective
against a number of human turnours. However, cyclophosphamide therapy is
accompanied by an array of toxic syndromes. Considerable interest has
been focused on the isolation of compounds from plants that reduce the dose
limiting toxicity of cyclophosphamide.
Unnikrishnan et a/ (1 990) reported that intraperitoneal administration
of garlic along with cyclophosphamide reduced the toxicity of the latter,
increasing the life span more than 70%. Garlic administration did not improve
lymphopenia produced by cyclophosphamide or liver alkaline phosphatase,
but there was significant reduction in liver glutamic pyruvate transaminase.
Moreover, it reduced the level of lipid peroxidation induced in liver by
cyclophosphamide. Administration of garlic did not interfere with tumour
reducing capacity of cyclophosphamide. Garlic extract contains several
sulphydryl groups and thiol compounds like alline, allicine etc, that may act
as effective free radical scavengers and promote repair.
Bhanumathy et a1 (1986) reported that the synthetic compound
mercaptopropionyl glycine (MPG) protected against the effect of
cyclophosphamide lethality, while retaining the anticancer properties of
cyclophosphamide. Cyclophosphamide induced leucocytopenia and
increased serum alkaline phosphatase and serum glutamate transaminase
levels were corrected by M PG treatment. The enhanced survival rate of
animals by MPG treatment may be due to the result of protection of the
gastrointestinal epithelium and haemopoietic organs of mice against
cyclophosphamide induced toxicity. Autoradiographic studies indicate MPG
in hibits mitosis temporarily during early hours. This may allow times for repair
processes to act before cyclophosphamide induced structural defects are
replicated. Therefore MPG is a useful adjuvant to cancer chemotherapy.
Varghese et a1 (1991) reported that the flowers and bark of Saraca
asoka (Caesalpinnaeae) had chemoprotective effect on cyclophosphamide-
induced toxicity in S-180 tumour bearing mice. Kumari (1 991 ) suggested that
the well known spice, clove, (Caryophyllus aromatics) had significant
modulatory effect on hepatic detoxification systems and bone marrow
genotoxicity in mice. Hus 1985) reported that a combination of Codonopsis
pilosula when used in combination with cyclophosphamide increased the
mean sutvival time and decreased the number of metastatic foci, though
when used singly it did no produce significant results. C.pilosula is widely
used in traditional Chinese medicine to increase immune function especially
macrophage activity (Mao and Zhou 1985). They suggested that Codonopsis
pilosula enhanced humoral immunity and cell mediated immunity of mice
immunosuppressed with cyclophosphamide, but had no activity on normal
mice. Methanol extract of different parts of Sfreblus asper did not show
antiturnour activity. However, the leaf and flower extracts when used with
cyclophosphamide potentiated the activity of the drug significantly (Ghanu et
a1 1 991 ). The extracts of Crotalaria and Senecio genera and ginger were also
reported to reduce cyclophospharnide toxicities (Nair 1991 a). Soudamini and
Kuttan (1990) reported that turmeric extracts as well as curcumin could
reduce the toxicity of cyclophosphamide. Some of the important medicinal
plants effective against cyclophosphamide toxicity are the following
(Table-5).
Table- 5
Plants reported to be effective against the toxicity of the anticancer
drug cyclophosphamide
Plant
A Ilium sa tivum
Crocus sativus
Codonopsis pilosula
Commiphora mukul
Nigella sa tiva
Saraca asoka
Streblus asper
Tinospora cordifolia
Viscum album
Action
increased life span
reduced liver glutamate
pyruvate transaminase
levels
increased life span
increased life span
stabilized cytological and
biochemical changes
increased life span
increased life span
increased life span
Reversed m ye lo
suppression
Reduced leucocytopenia
Reference
Unnikrishnan et a1
(1 990)
Nair et a1 (1 990)
Hus (1985)
Al Harbi et a1 (1 994)
Nair et a1 (1 990)
Varghese et a1 (1 991 )
G hanu et a1 (1 991 )
Sharma et a1 (1 995)
Kuttan and Kuttan
(1 992)
1.8 Infrared Spectroscopy
The field of "cancer detection" via infrared spectroscopic techniques
has had a somewhat rocky start, it is now apparent that cells and tissues
provide an immense amount of information on cellular composition, packing
of the cellular components, organ and cell architecture, metabolic processes
and absence or presence of disease. However, to reach this point, the field
required a new start, which was initiated three years ago by a number of
research groups (H. Fabian ef a1 1995; C.P. Schultz et a1 1998; P. Lasch et a1
1998; L. Chiriboga et a1 1998). Fourier transform infrared spectroscopy is
gaining recognition as a promising method in diagnostic, medicine and
biological studies. Both clinical and biological studies would benefit from the
spectroscopic technique that could quantify the biochemical composition of
specimens non-destructively and with minimal sample preparation and
handling.
Changes in infrared spectra between normal and neoplastic tissue
and cells may arise from a number of different sources. First, specific cellular
events such as changes in the genetic code or the synthesis of mutant
proteins occur in almost all forms of neoplasia. There is a little doubt that
these alterations will contribute to the spectral changes. However, it seem
unlikely that even a few mutant genes or proteins are sufficient to produce
the distinct neoplastic patterns so frequently and reproducibly encountered
thus far. Second, the changes may arise from secondary cellular events such
as increase (or decrease) in component classes (DNA, RNA, lipids, proteins,
carbohydrates) of particular interest is the role nucleic acids play in the
spectroscopic changes. Third, the observed spectral difference could arise
from different averaging processes, since the populations of cells at given
stages of the cell division cycle may vary between healthy and cancerous
samples. b
Fourier t ransforrn infrared (F TI R) spectroscopy, which is used to
measure the vibrational modes of the functional groups of molecules, is
sensitive to molecular structure, confirmation and environment. FTlR can be
used to detect biochemical changes and has become a powerful method for
probing the structure and the interaction of biomacromolecules such as DNA,
RNA, proteins, carbohydrates, lipids in biological tissues and cells (M.
Jackson et a/ 1993; P.T.T. Wong et a1 1993; Wong et a1 1992.)
1.8.1 Spectroscopy of major cellular components
Human cells come in a variety of shape and size. They may range
from a configuration that is about 10 to 15pm on edge, and nearly cubic in
shape, to stratified (flattened) morphologically, up to 60pm in diameter and
5-1 0pm thick, depending on the organ of origin. By dry weight, cells consist
of about 60% protein and 25% nucleic acids, with the rest from other
components (carbohydrates, phospholipids and others). In most healthy
cells, the RNAIDNA ratio is about five. This composition may vary depending
on the organ, the cell division cycle and other factors.
1.8.2 Infrared Absorption Spectra of Protein and Nucleic acids
The average protein spectra found inside cells are dominated by the
amide I band at - 1650cm-' that is primarily associated with the stretching
motion of the C=O group (Fig1 A). This peak is sensitive to the environment
of the peptide linkage and depends on the protein's overall secondary
structure. The amide II vibration, mainly a coupled C-N stretching and a CNH
deformation coordinate, occurs at 1 530cm-' . Weaker protein vibrations
include the amide Ill peak (coupled C-HIN-H deformation) at 1245cm-' and a
number of side chain vibrations in the 131 0, 1390 and 1450cm-' range
(Diem M 1993).
The infrared absorption spectra of RNA and DNA also depend on the
state of hydration and its secondary structures. They exhibit absorption
peaks between 1580 and 1700cm-' due to the aromatic base breathing and
C=O stretching vibrations. The ionized PO2 and ribose groups exhibit a triad
of peaks that occur in DNA at 1071, 1084 and 1095 cm" with nearly equal
intensities. DNA peaks are also observed at 965 and 1245 cm"
(the phosphodi&ter vibration) (Diem M 1993).
1.9 Rationale for the selection of the plant of this study
The plant used in this study is Barringtonia racemosa Roxb. It was
directly brought to my notice that the Adivasis or tribals in the Wayanad
district of Kerala are using this plant for the treatment of various cancer like
diseases with success. An extensive literature survey showed that
B.racemosa seed had not been investigated for its antitumour activity.
Accordingly, the seeds were selected for investigation.
Barringtonia racemosa Roxb: It is normally found on the landward edge of
wet tropical mangrove forest, often growing upstream in rivers. They may
grow to 20m tall. It comes under the family Lecythidaceae. The leaves can
be up to 40cm long and 15cm wide. They are pointed at the tip, have slightly
toothed edges and very pronounced veins. Flowers are arranged in long
spikes coming out of the center of leaf groups. The fruit is egg shaped and
bark is gray and generally smooth.
1.9.1 Properties and Uses of B.racemosa seed
The different parts of the plant are used for the treatment of various
diseases. The root is used as de-obstruent; fruit for curing cough, asthma
and diarrhea; Kerneal is used for cure on jaundice (taken with milk) and bark
is used as an insecticide. B.racemosa possess anti-bacterial activity
(Schimul Khan et a1 2001). The powdered seed is irritant to the nasal cavity
and produce sneezing (Watt J M et a1 1962).
1.9.2 Chemistry and Pharmacology
A new saponin-Barrington in-composed of rhamnose and
barringtogenol, M.P. 2 9 0 ~ ~ ~ from the fruit of the plant. (Bull. Res. Inst.
Kerala.Se a 1959, 6,l5. Chem. Abstr. 1960, 54, 16746 f).
The survey of literature pertaining to this plant did not show any
attempt to investigate this plant as a source of medicine for cancer.
However, some' investigations were carried out regarding its medicinal
properties. This has resulted in taking an active interest in this plant
particularly since it is being used by tribals of Kearla with remarkable
success. Hence, we thought it is interesting to investigate in detail this plant
as a source of medicine for the cancer treatment. Hence, the present
investigation was carried out.
1 .I0 Aims and Objectives
The study is to be carried out to understand the effectiveness f the
plant as antiturnour drug. The following aspect of the plant extract is to be
carried out to access the potential tumour reducing and tumour preventing
activities.
1. To study the activity of the plant by cytotoxic experiments on tumour
cell lines DLA and EAC. The different parts of the plant are to be
extracted and the cytotoxic property to be investigated.
2. The effect of various solvents on the extracts and isolation of active
fraction from the most active part of the plant.
3. The cytotoxicity studies are to be carried out by using the active
extract against DLA and EAC cell line and find out the minimum
concentration required for 50% cytotoxicity.
4. Toxicity studies are to be carried out as per protocol system to find
out where there is any toxicity to normal cells and if so acess the LDso
concentration of the drug.
5. Partial purification of the active ingredient.
6. Tumour prevention and tumour reducing property of the purified
fraction in experimental tumour production using DLA and EAC in
Swiss albino mice. Thus, investigation is to be carried out taking in to
consideration of the property or tumour prevention properties in
experimentally induced ascities.
7. Similar experiments have to be carried out by studying solid tumour
production -in the hind leg of mice.
have to be carried out in
31
normal
and tumour bearing mice during the administration of Vincristine or
Cyclophospharnide. The effect of the drug in reducing toxicity during
the administration of Vincristine or Cyclophosphamide.
9. The changes that can takes place during the development of solid
turnour and its possible reduction by administration of the drug can be
investigated by noting the changes in solid tumour formed in the
control and experimental animals by making use of spectroscopic
analysis of the tumour. This may gives some findings as additional
evidence for the tumour reducing properties of the drug.
10. The effect of the drug on different human cell lines in culture has to be
investigated.
11. Apoptosis is a physiological mechanism in the body and it is
interesting to study the effect of the drug to increase apoptosis
thereby reducing tumour production. This can be carried out at a non-
toxic level so that this could
12. It is interesting that howfar the drug can affect the chromosome in the
cell of normal and experimental cancer tissue. This may give a lead to
making of the drug.
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