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.