INTRODUCTION

1.1.Cancer

Cancer is a class of diseases or disorders characterized by uncontrolled division of

cells and the ability of these cells to spread, either by direct growth into adjacent tissue

through invasion, or by implantation into distant sites by metastasis (where cancer cells

are transported through the bloodstream or lymphatic system).

Cancer arises from cells in the body which were once normal cells. In the growth

of normal cells, a finely controlled balance exists between growth- promoting and

growth-restraining signals such that proliferation takes place only when required. This

order is tilted only when more cells are required such as in wound healing. In this

situation differentiation takes place in an orderly manner and proliferation ceases when

no longer required. However, in tumour cells this process is disrupted, cell proliferation

happens continuously and loss of differentiation can occur. The normal process of

programmed cell death may cease to operate These cells which are now “transformed” to

grow and divide and keep dividing in an uncontrolled manner. They differ very subtly at

first compared to the cells of the normal tissue from which they originate

A tumor or tumour is the name for a neoplasm or a solid lesion formed by an

abnormal growth of cells (termed neoplastic) which looks like a swelling. A tumor can be

benign, pre-malignant or malignant, whereas cancer is by definition malignant

Two types of tumors exist, benign and malignant. Benign tumors are not

cancerous. They can usually be removed by surgery and generally don't grow back The

cells in benign tumors don't spread and it is rare for a benign tumor to be life

threateningMalignant tumors, on the other hand, are cancerous. The cells in them are

abnormal and divide randomly and chaotically. The cells behave aggressively and attack

the tissue around them.

They also can jump away from the malignant tumor and enter the bloodstream or

lymphatic system to form new tumors in other parts of the body. This type of spread is

known as metastasis.

1.2. Origins of Cancer

All cancers begin in cells, the body's basic unit of life. To understand cancer, it's

helpful to know what happens when normal cells become cancer cells. The body is

made up of many types of cells. These cells grow and divide in a controlled way to

produce more cells as they are needed to keep the body healthy. When cells become old

or damaged, they die and are replaced with new cells.

However, sometimes this orderly process goes wrong. The genetic material

(DNA) of a cell can become damaged or changed, producing mutations that affect normal

cell growth and division. When this happens, cells do not die when they should and new

cells form when the body does not need them. The extra cells may form a mass of tissue

called a tumor

Fig 1:Difference between Normal and Cancer Cell Growth

* Benign tumors aren't cancerous. They can often be removed, and, in most cases, they

do not come back. Cells in benign tumors do not spread to other parts of the body.

* Malignant tumors are cancerous. Cells in these tumors can invade nearby tissues and

spread to other parts of the body. The spread of cancer from one part of the body to

another is called metastasis.

Fig 2:Benign and malignant tumors

.1.3. Classification:

Cancers are classified by the type of cell that resembles the tumor and, therefore, the

tissue presumed to be the origin of the tumor. These are the histology and the location,

respectively. Examples of general categories include:

Carcinoma: Malignant tumors derived from epithelial cells. This group

represents the most common cancers, including the common forms of breast,

prostate, lung and colon cancer.

Sarcoma: Malignant tumors derived from connective tissue, or mesenchymal

cells.

Lymphoma and leukemia: Malignancies derived from hematopoietic (blood-

forming) cells

Germ cell tumor: Tumors derived from totipotent cells. In adults most often

found in the testicle and ovary; in fetuses, babies, and young children most often

found on the body midline, particularly at the tip of the tailbone; in horses most

often found at the poll (base of the skull).

Blastic tumor or blastoma: A tumor (usually malignant) which resembles an

immature or embryonic tissue. Many of these tumors are most common in

children.

1.4.MORPHOLOGY OF CANCER CELLS

● Larger than normal cells

● Show nuclear and cellular polymorphism

● Hyperchromatism, alteed nuclear, cytoplasmic ratio, abundant mitosis

● Often grow over one another and form multilayers.

● Diminished adhesion

1.5.BIOCHEMICAL CHANGES

● Increased synthesis of DNA and RNA

● Show increased rate of glycolysis

● Show alterations of permeablity and memberane charge

● Changes in composition of glycoproteins and glycosphingolipids on cell surfaces

● Apperarance of new antigens and loss of certain antigens.

1.6.Carcinogen

Carcinogens are agents that causes cancer or increase the risk of getting cancer by

altering cellular metabolism or damaging DNA directly in cells, which interferes with

biological processes, and induces the uncontrolled, malignant division, ultimately leading

to the formation of cancer tumors. Carcinogenesis can be actively caused by chemicals,

radiation, and infectious biological agents. The action of specific carcinogenic agents

depends upon the stages of cancer development such as initiation, promotion, and

progression. The mechanism of the induction of carcinogenesis may be due to alteration

in the genomic structure. The final stage of carcinogenesis, i.e., progression, may occur

spontaneously enhanced by the formation and propagation of genetic errors. In addition,

chemical and viral agents that lack the capacity for initiation and promotion may actively

convert the cells in the stage of promotion to the stage of progression

Fig 3:Mechanisms of carcinogenesis

1.7.Molecular Mechanisms of Cancer:

Fig 4:Various Stages of Cell Cycle

Cancer is a disease that begins in the cells of the body. In normal situations, the

cells grow and divide as the body needs them. No more, no less. This orderly process is

disturbed when new cells form that the body was not needed and old cells don't die when

they should. These extra cells lump together to form a growth called tumor.

Cancers result from a series (progression) of gene mutations that typically involve

two categories of function: promotion of cell division and inactivation of cell cycle

suppression. Proto-oncogenes are normal genes that promote cell growth and mitosis,

whereas tumor suppressor genes discourage cell growth. Proto-oncogenes can be mutated

by carcinogenic agents to become oncogenes. Oncogenes produce excessive levels of

growth promoting proteins

Tumor suppressor gene products typified by p53 are frequently transcription

factors that suppress mitosis and cell growth to allow for DNA repair. Nearly half of all

cancers involve altered p53 genes. Other suppressor genes include Rb (retinoblastoma

family), APC (adenomatous polyposis coli), SMAD4, TP53, p16/CDKN2A and BRCA

(breast cancer susceptibility protein) types 1 and 2.

Cancer results from cumulative mutations of proto-oncogenes and suppressor

genes which together allow the unregulated growth of cells. Oncogenes are typically

dominant because they provide gain-of-function, whereas suppressor genes are recessive.

They contain loss-of function mutations. Both copies of a suppressor gene need to mutate

to cause loss-of-suppressor function. Only one copy of a proto-oncogene needs to mutate

for gain-of-function. Mutations of tumor suppressor genes can be inherited.

Over time malignant cells can self-select for characteristics that make them more

malignant: ability to avoid apoptosis; immortalization due to over expression of

telomerase; growth-factor self-sufficiency and resistance to anti-growth factors; increased

cell division; altered differentiation; loss of contact inhibition, become metastatic; and

able to promote angiogenesis.

1.8.Symptoms of cancer:

Symptoms of cancer metastasis depend on the location of the tumor.

Local symptoms: unusual lumps or swelling (tumor), hemorrhage (bleeding), pain

and ulceration. Compression of surrounding tissues may cause symptoms such as

jaundice).

Symptoms of metastasis (spreading): enlarged lymph nodes, cough and

hemoptysis, hepatomegaly, bone pain, fracture of affected bones and neurological

symptoms. Although advanced cancer may cause pain, it is often not the first

symptom.

Systemic symptoms: weight loss, poor appetite, fatigue and cachexia excessive

sweating anemia and specific paraneoplastic phenomena, i.e. specific conditions

that are due to an active cancer, such as thrombosis or hormonal changes.

1.9.Existing lines of treatment:

Conventional cancer treatment can include surgery, radiation, chemotherapy, hormone

therapy, and biological therapy , mono clonel antibody therapy.

Radiation therapy:

Radiation therapy is the use of ionizng radiation to kill cancer cells and shrink

tumors.Radiation therapy can be administered externally via external beam radiotherapy

(EBRT) or internally via Brach therapy. The effects of radiation therapy are localized and

confined to the region being treated.

Chemotherapy:

Chemotherapy is the treatment of cancer with drugs (anticancer drugs) that can

destroy cancer cells. It interferes with cell division in various possible ways, e.g. with the

duplication of the DNA or the separation of newly formed chromosomes. Most forms of

chemotherapy target all rapidly dividing cells and are not specific for cancer cells hence

the chemotherapy has the potential to harm healthy tissue especially those tissues that

have a high replacement rate. These cells usually repair themselves after chemotherapy.

Chemotherapy is a treatment based on action of several agents such as:

Mitoxantrone, Vinorelbine, Docetaxel, Estramustine or others. Basically, chemotherapy

agents kill cancer cells that are actively multiplying

The general disadvantage of chemotherapy, no matter of type of cancer, is the

drugs cannot discriminate between fast-growing cells and kills all cells whether they are

part of controlled or uncontrolled process..

Surgery:

Cancer can be cured if entirely removed by surgery, but this is not always

possible. When the cancer has metastasized to other sides in the body prior to surgery,

complete surgical excision is usually impossible.

Immunotherapy:

More contemporary methods for generating non specific immune response against

tumor include intraversial BCG immunotherapy for superficial bladder cancer, and use of

interferon and interleukin. Vaccines to generate non-specific immune responses are

subject of intensive research for a number of tumors notably malignant melanoma and

renal cell carcinoma.

Hormonal suppression:

The growth of some cancers can be inhibited by providing or blocking certain

hormones. Common examples of hormone-sensitive tumors certain types of breast and

prostate cancers.

Photodynamic Therapy:

Photodynamic Therapy is generally a non-invasive treatment using a combination

of light and a photosensitive drug. Photodynamic Therapy, also known as PDT, uses

photosensitive drugs (such as 5-ALA, Foscan, Metvix, Tookad, WST09, WST11,

Photofrin and Visudyne) which are triggered by light of a specific wavelength.

Telomerase therapy:

Most malignant cells rely on the activity of the protein telomerase for their

immortality, it has been proposed that a drug which inactivates telomerase might be

effective against a broad spectrum of malignancies. At the same time, most healthy

tissues in the body express little if any telomerase, and would function normally in its

absence.

1.10.Plant source as tumor remedy

Plant products have been used for centuries as medicines. Today in most

prevalent treatments, with recipes handed down from generation. They are available and

are less costly then allopathic medicine, practitioners are available, and there is generally

a more culturally sensitive attitude on the part of these practitioners. Each herb may hold

active chemicals. Many studies revealed reduced incidences of many common forms of

cancer if treated with herbs, which may contain phytochemicals that can modulate gene

expression inhibit carcinogenesis via multiple pathways.

Many plants synthesize substances that are useful for the maintenance of health

in humans and other animals. These include aromatic substances, most of which are

phenols or their oxygen-substituted derivatives such as Tannin. Many are secondary

metabolites, of which at least 12,000 have been isolated – a number that is estimated is to

be less than 10% of the total. In many cases, these substances (particular the alkaloids)

serve as plant defense mechanisms against predation by microorganisms, insects, and

herbivores. Many of the herbs and spices used by humans as season food also yield useful

therapeutic compounds with only a few exceptions, most herbal treatment have not been

tested for safety and efficacy utilizing scientific parameters. Proper clinical trials were

also not conduced.

In the present dissertation a commonly available plant belonging to Verbinaceae

is selected and screened for the its anticancer potential. This plant source is used by

Ayurvedic practitioners for various preparation that controls several oilments. This is

known as “Ganakasika” botanically equated as Premna integrifolia Linn. Ethanolic

extract of the aerial portion of this traditional drug source is scientifically validated for its

anticancer potential.

REVIEW OF LITERATURE

Two novel tritrepene acids, Frculontonic acid A(1) and B(2) as well as the extract

of leaves, stems and twigs of Manihot esculenta studied for cytotoxic activity .The

structures of two new compounds were established on the basis of 1D and 2D NMR

spectroscopic data interpretation and chemicals conversions. The two new compounds

showed moderate cytoxicity against the A2780 human ovarian cancer cell lines.

(Chaturvedula VSP etal., 2004)

Melicarpine, Semecarpine and (+)-8-methoxyplanty desmine, together with

known flavone,ayanin were isolated from the leaves of Melicope somecarpifolia All the

compounds along with 15 compounds isolated previously from the same plants were

tested against P-385,HA-29 and A 549 cell lines in vitro . Among them confusameline

was found to be most cytotoxic isolate and exhibited most potent activity (ED50=0.03

(μg/ml) against P-388 cell lines. (Chenm JJ etal., 2004)

Seven known pentacyclic triterpenes and one steroid were isolated from

Euphorbia lagascae methanolic extract and identified by physical and spectroscopic

methods. These compounds together with eleven terpenoids previously isoalated from

Euphorbia lagascae and E.tuckeyana were tested for their apoptosis induction activities

by flow cytometry  on L6178 human MDR1 gene-transfected lymphoma and result

showed moderate apoptosis induction effects.(Duarete N etal., 2009)

The petroleum ether fraction of Buxus microphyla was subjected column

chromatography. Eight components were identified namely,butulin, (1)beta-sitosterol,

(2)stigmasterol, (3)dibutyl phthalate, (4)3beta, 30-dihydroxy-lup-20(29)ene,

(5)daucosterol.  The compound 5 should potent anticancer effects against KB cells (Dai

ZK etal., 2009)

A flavanone has been isolated from Bauhinia variegate and its structure was

identified by colour reactions and spectral analysis. The isolated flavanone was tested for

cytotoxic activity against 57 human tumour lines. Respectively leukemia, non-small cell

lung, colon,central nervous system maianoma, ovarian,renal,prostate and breast cancers.

The results showed that the flavanone showed cytotoxic activity against human tumour

cell lines.(Rajkapoor B etal.,2009)

The study aimed to at evaluate the cytotoxic mechanism of Calotropin on human

chronic myeloid leukemia K562 cells. Calotropin inhibited the growth of K562 cells in a

time and dose-dependent manner by G(2)/M phae arrest. It up regulated the expression of

p27 leading to this arrest by down regulating theG2/M regulatory proteins cyclins A and

B,and by up regulating the cdk inhibitor.p27furthermore,it down regulated anti-apoptotic

signalling (XTAP and surviving) and survival pathways (P-Akt and NFKappaB) leading

to caspase-3 activation which resulted in the induction of apoptosis.(Wang SC etal.,

2009)

The Invivo efficacy and mechanisms of action of oral administrations of a

standardized extract of W.chinensis were analysed in animals bearing  a  subcutaneous

orthiopic prostate cancer xenograft. Exposure of prostate cancer cells to W.chinensis

extract induced apoptosis pathway. Selectively in androgen receptor (AR) positive

prostate cancer cells and shifted the propotion in each phase of cell cycle towards(2)-M

phase in AR negative prostate cancer cells. (Tsai CH etal.,2009)

Gingerol, a natural components of ginger exhibits antiinflamatory and  anti

tumorigenic activities. Despite  its potential efficacy in cancer, the mechanisms by which

-gingerol exerts its chemopeventive effects remains unclear. The leukotriene A(4)

hydrolase (LIA(4)H) protein regarded as a relevant target  for cancer therapy. In silico

prediction using a reverse –doking approach revealed that  LTA(4) H might be a potential

target of gingerol. Gingerol suppresse anchorage independent cancer cell growth by

inhibiting LTA(4)H activity in HCT 116colorectal cancer cells. (Jeong CH etal., 2009)

study discovered on antitumour bioactive compounds from That indigenous

plants. Cytotoxicity of the extracts was evaluated against two cancer cell lines.Human

amelanotic metanoma) and humancervical carcinoma (Hela)by the SRB assay.

Antihelmintic and anticancer activities were evaluated by the inhibiting concentration at

50% death.(IC(50) and the selectivity index(ST) relative to human fibrbiasis. Strong

cytotoxicity was shown by the methylene chloride extract of Michelia champaca bark

and the methanol extract of Carcama longarhizome against C32 and Hela cell lines

respectively.(Atjanasuppat K etal., 2009).

Isoliquiritigenin (4,2,4-trihytrochlcone,ISL)is a natural pigment  with a simple

chalcone structures. The cell growth inhibition achieved by ISL treatment resulted in

programmed cell death manner, with an IC50 of 10.50µg/ml.(Hso yl etal., 2005)

The anti-tumour activity of the diethyl extract of zingiber  zerumber was

measured in cultured P-388DI cells and on animal model of P03881bearing CDF mice.

The results showed DNA fragmentation in p-38801 bearing CDF1 mice at a dosage of

5mg/kg  body weight .(Huang GC etal., 2005)

Casuarinin  a hydroluzable tannin isolated from the bark of Terminalia arjuna was

investigated for its antiproliferative activity in human breast adenocarinoma  MCF-7

cells. The result showed that casuarinin indues the apoptosis in MCF-7 cells.(kuo PL 

etal., 2005)

Evodiamine isolated from  Evodia rutaecarpa  showed Antiproliferation and 

apotosis  inducing activity  against  human colorectal carcinoma cells(colo-205).(Yang

ZG etal.,2009)

The methanolic extract of leaves of Cassia tora (CTME) showed potent lipid

peroxidaxlon inhibitory activity as well as showed potent antiproliferative and apoptosis

inducing activity against HeLa cell lines.( Rejiya CS etal., 2009).

Vijayan  etal 2004 reported the in vitro cytotoxicity and anti-tumour  properties of

Hypericum  mysorense and Hypericum  patulum. The methonalic extracts in the

H.mysorense and H.patullum was tested against HEp-2 cell lines the results that the

both plant extraction showed moderate cytotoxic effect against HeLa cell lines.

Bioassay –direced fractionation of Saussurea lappa lead to the isolation of a

novel lappadilactone and some sequiterpene lactones as cytotoxic principle against

selected human cancer cell lines. lappadilactone, dehydro-costuslactone and cortunolide

exhibited the most potent cytotoxicity with CD50 values in the range 1:6-3.5 µg/ml in

dose and time –dependent manners. The cytotoxicities were not specific and showed

similar activities against HepG2, OVGPR-3 and HeLa cell lines. (Syu CM etal., 2009)

The anti metastatic effects of Magonolia isolated from Magnolia oborata were

evaluated by an experimental liver and spleen metastasis model using 1.5178-m.25

lymphoma and Melanoma. Administration (i.p) of 2 or 10 mg/kg of magnolia

significantly suppressed liver and spleen metastasis. These data suggest that magnolia

possess strong anti metastatic ability and that it may be a lead compound for drug

development. (Ikeda k etal., 2004)

Aqueous and organic extracts of Asteraceae (compositae) collected from the state

of Rio Grande do sul, Brazil were tested in vitro or cytotoxic activity against human solid

tumour cell lines. Twenty- five species, 125 extracts in total, were screened against HT29

human colonadenocarcinoma cells and Ncl-H460 human non- small cell lung cancer

cells. Extracts Bacchoris cordifolia,B.ochracea, Eupatorium macrocephalum,

E.pedunculosum and Stenachaenium niedeli all produced IC 50 values below 5 mg / dl.

(Monks NR etal., 2004)

Gleditsioside E.a triterpene saponin isolated from Gleditsia sinensis, showed

significant cytotoxicity against BEL-7402, BGC-823, HELA,HL-60 and MCF-7 cell

lines.”Induction of apoptosis and G2/m cell cycle arrestare the mode of action of

Gledisioside E .(Zhong L etal., 2003)

The methonalic extract of pereshipa bleo showed cytotoxic activity against T-47D

cell with Ec 50 of 2.0mg/ml .the extract induces the DNA transformation ,chormarin

Marigination which results in the activation of apotosis in T-47D cells(Tan ML etal.,

2005)

The effect of six xanthones from Garcinia mangstana were examined on the cell

growth inhibition of human leukemia cell line HL60 All xanthones displayed growth –

inhibitory effects. Among them alpha mangostion showed complete inhibition at 10µ/m

through the induction of apotosis. .(Matsumoto K etal., 2005)

The flavanones 5,6-dihydroxy-7,3-4-trimethoxy flavones,5,6,4-trihydroxy-7,3-

dimethoxy flavone, 5-hydroxy-3-4 6,7-tetramethoxy flavones, 5,7,3-trihydroxy-6,4,5-

trimetoxy flavone, ladanein and hispidulin were isolated from the methenolic extract of

the Cerial parts of Artemisia argyi and structure compounds were elucidated on the basis

of their structural data. These flavanones inhibited farnesyl protein transferees with IC50

values of 25-200 µg/ml. compound 2 inhibited proliferation of a couple of tumor cell

lines ( Leo JM etal., 2003)

The 7,8 –dihytroxy flavanone isolated from the seeds of Alpinia katsumadai was

found to have an in vitro cytotoxic effect against A 549 (a human cancer cell lines) and k-

562 (a human leukemia cell line. (Hahm ER etal., 2004).

Poochendura pattai a traditional drug which is botanically equivalted as

Plectranthus urticifolius Hook. F. is screened for its antitumour potential, and gave

encouraging results (Brindha P etal.,2000)

Embelin a naturally occurring benzoquinone isolated from Embelia ribes showed

significant antitumor activity by increasing the life span and reversing the elevated levels

glycorportein in tumor bearing rats. (Chitra M etal., 2004)

In EAC tumor induced mice the methanol extract of Mucuna pruriens at the

doses of 125mg and 250mg/Kg. bw significantly inhibited the tumor volume, Packed cell

volume and cell count and brought back the hematological parameters ( Rajeswar etal.,

2005)

Ursolic acid pentacyclin triterpene component was screened for anti cancer

potential against endometrial cancer cell line SNG – II. It was found that the ursolic acid

strongly inhibited the SNG II cell lines in dose and time dependent manner, Mechanism

behind this was found by activity of apoptotic pathway which decrease the BCL-2 protein

20(anti apoptotic factor) and increased BAX protein expression ( Apototic factor)

(Yumiko Achiwa etal., 2005)

Withaferin A isolated from Withania somnifera showed marked tumor inhibitory

activity in carcinoma Nasopharynx, Ehrlich Ascites carcinoma, Sarcoma 180, Sarcoma E

0771, and mamaray adeno carcinoma cell lines in animal models. ( Chakrabortui etal.,

1974)

Administration of Black Tea to EAC bearing Swiss albino mice caused significant

decrease in the tumor cell count and increased life span in dose dependent manner,

( Arindan Bhattacharyya etal., 2003)

From the literature we found that the plant drugs plays a significant role in the

development of anticancer drugs. Hence in the present study a common plant “Munnai”

botanically equated as Premna Integrifolia Linn. Belonging to the family Verbenaceae

was selected and the ethanolic extract of the plant material was screened for anticancer

activity against Ehrlich Ascites carcinoma cell lines.

AIM AND OBJECTIVES

Cancer is one of the deadliest as well as induced disease characterized by

uncontrolled and unwanted growth of cells leading to the dysfunction of the organs

where it proliferates. Many treatment modalities are available for cancer at present but

they are always associated with side effects. Traditional plants might provide useful

sources for developing new anticancer drugs and could be a good alternate to existing

lines of cancer therapies.

Hence need of the hour is to develop an anticancer drug which is cost effective,

human compatible with proven anticancer effect. Based on the literature review, present

investigation aims at to identify and develop a novel anticancer herbal drug.

Premna integrifolia Linn. belonging to the family Verbenaceae was selected

and the ethanolic extract of the plant was screened for anticancer potentials against

Ehrlich Ascites Carcinoma cell lines. Studies were also carried out from standardization

point of you.

The main objectives of the present study are:

for the selected plant drug. To determine the Chemical Standards

To carry out phytochemical screeing for various extracts.

To conduct In-Vitro Cytotoxic studies against Ehrlich Ascites Carcinoma

cell lines.

To conduct in-depth out In-Vivo anticancer studies against Ehrlich Ascites

Carcinoma cell lines

Table 7-Cytotoxic Effect Of EEPI on EAC cells (Trypan Blue method)

Concentration of Viable cells Viable cells Death cells Death cells

EEPI

(μg/ml)

(%) (%)

Control 91 95.79 4 4.21

50 95 79.32 30 20.68

100 78 76.83 37 32.17

250 76 59.4 52 40.60

500 79.75 45.94 93 53.06

Dead cell = Accepts trypan blue dye

Viable cell = Not accepting trypan blue dye

Fig 6:SHORT TIME INVITRO CYTOTOCITY STUDY

4.21

20.68

32.17

40.6

53.06

0

10

20

30

40

50

60

Control 50 100 250 500

μg/ml

per

cen

tag

e o

f ce

ll d

eath

In –vitro cytotoxic effect of the Ethanolic extract of Premna integrifolia Linn.

.(EEPI) against EAC cell lines was Tabulated and graphically represented in (Table 7 &

Fig 6). At minimum concentration (50 µg/ml) showed 20.68% cell death where as the

higher concentration (500 µg/ml) showed 53.06% of cell death. The percentage of

cytotoxicity was found to increase with increase in the concentration of EEPI.

Table 8-Effect of EEPI on survival time of the Tumor bearing mice

Particulars Mean survival time Increase in life span(%)

(MST) (days) (ELS)

EAC control 19±1.1

EAC control + EEPI

(100mg/kgbw.)24±1.37 26.31

EAC control + EEPI

(200mg/kgbw.)29±2.71* 52.63*

EAC control + 5FU

(20mg/kg bw)31±1.59* 63.15*

Values are Mean ± S.E.M., n=6

*p<0.01statistically significant when compared to EAC control group

EEPI: Ethanolic Extract of Premna integrifolia

EAC: Ehrlich Ascites Carcinoma

Fig 7: Effect of EEPI on survival time of the Tumor bearing mice

0

26.31

52.63

63.15

0

10

20

30

40

50

60

70

Group II GroupIII Group IV Group V

Incr

easi

ng

in

lif

e sp

an%

Table8 indicates the increase in the life span of tumor bearing animals. On

treatment with various concentration of EEPI. The animals which received the 200mg/kg

body weight of EEPI showed 52.63% of increase in the life span of tumor bearing

animals (Fig 7). The standard drug 5-flurouracil increased life span up to 63.15%

Table 9 –Effect of EEPI treatment on tumor growth

Values are Mean ± S.D, n = 6

*p<0.05statistically significant when compared to Group II

Group 1: Normal control

Group 2: EAC control

Group 3: EAC + 100 mg/kg bw. of EEPI.

Group 4: EAC + 200 mg/kg bw. of EEPI.

Group 5: EAC + 20 mg/kg bw. of 5-fluoro uracil.

Fig 8: Effect of EEPI treatment on tumor growth

particularsTumor

volume(ml)

Viable cell 10 10 cells/L

Non-viable cells

1010 cells/L

Group I - - -

Group II 1.5±0.32 4.7±0.40 0.32±0.07

Group III 1.1±0.41 4.2±0.49 0.9±0.10

Group IV 0.7±0.27* 2.1±0.21* 1.4±0.13*

Group V 0.5±0.36* 2.7±0.34* 1.9±0.21*

1.5

1.1

0.7

0.5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Group I Group II Group III Group IV Group V

Tu

mo

ur

vo

lum

e (

ml)

The EAC inoculated mice showed increased Ascites fluid volume (1.5±0.32) and

increased viable cell counts (Table 9). On oral treatment with two different concentration

of EEPI administration for 14 days decreased the Ascites fluid volume group III

(1.1±0.41) and group IV (0.7±0.27) (Table )decreased viable cells count and increased

non- viable cells counts of tumor bearing animals (Fig 8)

TABLE 10 -Effect of EEPI on Hematological parameters  

Particulars Hb

(g %)

RBC count

106 cells/mm3

WBC

103 cells/mm3

Group I 15.3±1.4 5.9±0.3 6.7±0.8

Group II 11.1±1.4 4.3±0.5 9.3±0.5

Group III 13.3±.0.57 4.8±0.3 8.6±0.3

Group IV 14.3±0.48* 5.2±0.3* 7.3±0.3*-

Group V 15.1±0.35* 5.4±0.2* 6.7±0.7*

 

Values are Mean ± S.D, n = 6 

*p<0.05statistically significant when compared to Group II 

Group 1: Normal control

Group 2: EAC control

Group 3: EAC + 100 mg/kg bw. of  EEPI.

Group 4: EAC + 200 mg/kg bw. of  EEPI.

Group 5: EAC + 20   mg/kg bw. of 5-fluoro uracil.      

DISCUSSION

Cancer is one of the second largest killer disease in the world, and more than 11

million people were diagnosed as cancer patient every year. Death rate due to cancer is keeps on

increasing. However most of the modern therapies available for cancer treatment at present are

capable of producing serious side effects.

Need of the hour is to find a safe, cost effective and efficacious drug for this

deadly disease. In the present study with a view to develop a safe a plant anti cancer herbal drug

botanically equated as Premna integrifolia Linn. belonging to the family Verbenaceae was

selected and screened anticancer potential against Ehrlich Ascites Carcinoma cell lines,

employing both In-Vitro and In-Vivo methods. As standardization plays a vital role in herbal

drug development, in the present study physico and phyto chemical standards were determined

for the plant drug selected for the study.

TEST FOR IDENTITY, PURITY AND STRENGTH:

(1) Foreign matter(%) - Not more then 2%

(2) Total ash content (%) - Not more then 4%

(3) Water soluble ash (%) - Not more then 3%

(4) Acid insoluble ash (%) - Not more then 1%

SUCCESSIVE EXTRACTIVE VALUES:

(1) Hexane (%) - Not less then 2%

(2) Chloroform(%) - Not less then 2%

(3) Ethyl acetate(%) - Not less then 11%

SOLUBILITY:

(1) Alcohol (%) - Not less then 9%

(2) Water(%) - Not less then 18%

Fluorescence analysis of the plant drug revealed the presence of Brown,

Yellow and Green fluorescence in Day light.and UV light showed various shades of

green fluorescence (Table3 ). This indicates the presence of Alkaloids, Flavones, and

Steroids in the plant drug powder Under study.

Phytochemical screening of drug powders as well as various extracts

confurmed the presence of Alkaloids, Flavanones, Steroids, Coumarins, Lignin and

Quinones.

The organic constituents such as carbohydrates, protein and phenol were

found to be higher in the plant material. The phenolic content may be attributed to the

therapeutical potential of the plant drug under study.

The cytotoxic potential of Ethanolic Extract of Premna integrifolia

Linn. (EEPI) was tested against Ehrlich Ascites Carcinoma (EAC) cell lines using

Trypan Blue dye exclusion method. The preliminary experiment was carried out using

five different concentration of test drug. At the concentration 500 g /ml EEPI showed

53.06% of cell death whereas low concentration as much as 50 g/ml showed 20.68% of

cell death. (Table 7) As the part of the apoptosis precede the loss of membrane integrity,

cells were permeable to the Trypan Blue.

The reliable criteria for judging the value of any anticancer drug is

prolongation of life span of tumor bearing animals. (Clarkson BO etal.,1965) In the

present study EEPI increased the life span of tumor bearing animals up to 52.63%

(Table 8). Hence the test drug might be considered as a good anticancer drug.

The ascites fluid is essential for tumor growth because it constitutes the

essential nutritional source for tumor (Feccchi OD etal.,1990). The ascites fluid

volume will directly represent the tumor growth. In the presence study EEPI effectively

reduced the ascites fluid volume this clearly indicate that the test drug provides the direct

cytotoxic effect to the tumor cells or may inhibit the vascular permeability and cellular

migration.

In cancer therapy major problem encounted are (RBC) myelosuppression and

anemia. (Price  VE  etal.,1958).This may be due to the excess hemolysis. On treatment

the RBC and hemoglobin levels were reverted back to normalcy. This indicates that the

EEPI are capable of protecting the hemopoietic system. The increased WBC count of

tumor bearing mice are due to immune response against cancer cells. The criteria for

good anticancer drug is to reduce the WBC count. The present study EEPI treatment

reduced the WBC count. Hence the test drug may consider as good anticancer drug. 

            The lowered blood glucose hypoglycemia was reported in the EAC animals by

(Baliant  Z  1191). Administration of two dose levels of EEPI to the EAC animals

increased the blood glucose level. This indicated that the plant drug suppressed the solid

tumors by blocking the energy fuel of cancer cells. 

            The decreased serum protein content of EAC animals is due to the functional

impairment of hepatic tissue. On treatment protein levels found to be increased. This

indicated that the plant drug protect the hepatic tissue. 

            The induction of cancer resulted in the increased serum Cholesterol and

Triglycerides levels. This  may be due to increased lipolytic activity,  on treatment with

EEPI the serum cholesterol and Triglycerides were found to be decreased. This indicates

that the test drug may block the lipolysis and increased the lipogenesis. 

                  EAC inoculated mice showed increased amount of nucleic acid content such

as  DNA & RNA (Gkazer  RT  etal., 1971). This may be due to the increased cellular

proliferation and . On treatment with test drug the increased level of DNA & RNA were

restored to normal level. This might  be due to blocking of proliferation of tumor cells at

G1 phase of cell cycle and inhibiting the activity of polymerase enzymes. 

 

In the present study oral treatment of two different concentrations of EEPI

(100&200 mg/kg bw) to the EAC induced animals resulted in increased life

span ,reduced tumor growth and restoration of the biochemical ,hepatic marker

enzymes ,glycoprotein component and antioxident system.The data of the results

obtained in the present study were comparable with the result obtain on treatment with

the standard drug 5-FU and it was found that the dose level (200mg/kg bw) of EEPI

was found to be the effective dose.

SUMMARY AND CONCLUSION

Cancer is one of the deadliest genetic as well as induced diseases resulting due to

the defects in the genetic material. It is characterized by uncontrolled and unwanted

growth of cells. This is because of the loss of cell growth control mechanism caused by

various carcinogenic agents. Today modern medicine provides number of conventional

methods such as Radiation therapy, Chemotherapy, Hormonal suppression, and

Monoclonal antibody therapy. But these lines of treatments are known for their serious

side effects. Plants and plants derived products have been widely accepted as a good

therapeutic agents with lower side effects particularly in the management of cancer.

Based on the literature review, in the present dissertation a common traditional

plant source Premna integrifolia Linn, belonging to be verbenaceae family was selected

and ethanolic extract was screened for anticancer activity against Ehrlich Ascites

Carcinoma cell lines.

BIBLIOGRAPHY

Anonymous. The Ayurvedic pharmacopoeia of India. Government of India,

Ministry of Health and Family welfare, Department of Indian system of medicine and

Homeopathy, New Delhi. 2001; V(1); P 142-143

Arindam Bhattacharyya, Tathagata Choudhuri, Suman pal.2003.apoptogenic

effect of black tea on ehrlich’s ascites carsinoma cell, carcinogenesis,V 24;P 75-80.

Atjanasuppat K, Wongkhan W, Meenpowpan P, Kittakoop P and Spbhon

P.2009.In vitro screening for anthelmintic and antitumour activity of ethnomedicinal

plants from Thailand.J Ethnopharmacol,V 123(3); P 475-82.

Balint Z.1991. Lipolytic activity of in adipose tissue of mice bearing Ehrlich

Ascites Carcinoma. Cancer Biochem Biophys, V(12);P 45-50,

Brindha P, Sasikala, and Bhima Rao, Pharmacognostic studies on Coleus

Aromaticus. Benth. Indian Borage , B.M.E.B.R, Vol XII;P 17-31

Brindha.P, Anti - tumor activity poochendura pattai, Ancient Science of

Life,2000.V 20; P 82-84.

Burton K. (1956). A study of the conditions and mechanism of the

diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.

Biochem J V(62);P 315-32

Chakraborti sk De BK Bandyopadhyay T.1974. variation in the Antitumor

constituents of withania somnifera, In: Experientia ,V 30(8);P 852-853

Chase CR, and Pratt R.1949. J Am. Pharm. Assoc. Sci. Ed.;V(38);P 324.

Chaturvedula VSP, Schillins JK, malone s, wisse JH, werkhoven MCM, and

kingtom DGI.2004. New cytotoxic tritrepene acids from above ground parts of

Manihot esculenta form the suriname rainforest, Planta medica, V 69(3); P.271-

274.

Chen JJ, Duh CY, Huang HY, and Chen IS.2004. Furoquinoline alkaloids

and cytotoxic constituents from the leaves of Melicope semecarpifolia. Planta medica,

V 69(6); P.542-546.

Chitra M, Shyamala Devi CS,and Sukumar E.2004. Effect of embelin on

carbohydrate moietiesof glycoprotein in tumour-bearing rats, Journal of natural

Remedies, V 4(1);P 77-80.

Clarkson BD, Burchenal JH.1965.Preliminary screening of antineoplastic

drugs,Prog Clin Cancer,V(1);P 625-629,

Dacie JV, Lewis SM.1958. Practical hematology , 2nd ed., London: J and A

Churchill,P 38-48

Dai ZX, Liang J, Su XJ, Xu Q, and Zhang HQ.2009. To study the chemical

constituents from the petroleum ether fraction.Quilin medical college,V 32(7): P. 1062-4

Dishce Z, hettles LB.1948. Spectrophotometric micro method for tehr

dermination of Fucose, J.Biol.Chem,V(175);P 595-604.

Duarte N, Ramalhete C, Molnara J, and Ferreira MJ.2009. .Multidrug

resistance modulation and apotosis induction of cancer cells by terpenic compounds

isolated from Euphoria species. Anticancer Research ,Faculty university,V 29(11):

P.4467-72

El-Beshbishy HA. The effect of dimethyl dimethoxy biphenyl dicarboxylase

against tamoxifen-induced liver injury in rats. DDB use is curative or protective.

J.biochem. Mol.Biol., V(8);P 300-306

Feccchio D, Sirois P, Russo M, Janear S.1965.Studies on inflammatory

response induced by Ehrlcih tumor in mice peritoneal cavity. Inflammation.,V(14);P

125-131

Folin O and Wu H.1919.Determination of Glucose. J Biol Chem. 81

Foster LB and Dunn RT.1973. Stable reagents for determination of Serum

triglycerides by a colorimetric Hantzsch condensation method . Clin Chem V(196);P

338-340.

Fuji K, Node M, Sai M, Fujita E, Takeda S, Unemi N.1989. Terpenoids. LIII.

Antitumor activity of trichorabdals and related compounds, Chem Pharm Bull

(Tokyo),V 28;P 76-80.

Gamble JS.1997. Flora of Presidency of Madras, Botanical survey of India. Vol

II;. P 1096

Gkazer RI, Weber G.1971. Iodoacetate inhibition of lactate production and

lipid, Protein, ribonucleic acid and deoxyribonucleic acid synthesis in newborn rat brain

cortex sites. biochem Pharmacol, V(20);P :2121-2123.

Gothoskar SV, Ranadive KJ.1971.. Anticancer screening of SAN-AB, An

extract of marking nut Semicarpus anacardium. Indian J Exp Biol, V( 9);P 372-5.

Hahm ER, Pane S ,and Yang CH.2004. 7,8 –Di hydroxy flavanone ,as an

inhibition for Jus-Fos-DNA complex formation and its cytotoxic effect on cultured

human cancer cells. Natural product Research V,17(6); p.431-436,2003.

Halliwell B, Gutteridge JMC.1989. oxidants and carcinogenesis. In: Free

radicals in biology and medicine, 2nd ed. Oxoford: Oxford University Press, P 469-471.

Hso yl, Kuo PL, Lin LT, and Lin CC.2005. Isoliquiritigenin inhibits cell

proliferation and induces apoptosis in human hepatoma cells. Planta medica, V 71(2); P

130-134,

Huang GC,CHIEN TY, Chen LC, and wang CC.2005. Anti tumor  effects of

zerumbone from zingiber  zerumbet in p-388D,cells in vitro and in vivo .planta

medica, V 7(3); P 219-224.

Ikeda K, Sakai Y,and Nagase H.2004. Inhibitory effect of magnolol on tumour

metastasis in mice. Phytotheraphy Research.V 17(8);P 933-937.

Jeong CH, Bode AM,Pugliese A, Cho YY, and Kim HG.2009.(6) Gingerol

suppresses colon cancer growth by targeting leukotring leukotriene A4 hytrolase..Cancer

Res, V 69(13);P 5584-91.

King J.1965 .In : Practical Clinical Enzymology, Princeton MJ(Fol) Van D

Nostrand Company, London.P 363

275

Majumdar UK, Gupta M, and Maiti S.1997. Antitumor activity of

Hygrophila spinosa on Ehrlich Ascites Carcinoma and Sacroma 180 induced mice.

Indian J.Exp. Biol.V (35); P 473-477.

Malick CP, and Singh MB.1980. Estimation of Phenol. In: Plant Enzymology

and histoenzymology.. New Delhi. Kalyani Publishers; P 286.

Matsumoto K,Akao Y, Ksbasho E, ohguchi K, Ito T, Tanaka T, linuma

M,and Nozawa Y.2005.Induction of apotosis by xanthones from mangosteen in human

leukemia cell lines.Gift International Institute of Biotechnology,V 8(27);P 284-286.

Misra HP, Fridovich I.1972.The role of superoxide anion in the autooxidation of

epinephrine and a simple assay for SOD. J.Biol Chem, V(247);P 3170-3175.

Monks NR , Ferraz A, Bordignon S, Machado KR, Lima MFS, Rocha AB

Schnartsman.2004. In vitro cytotoxicity of extracts from Brazillian Asteraceae.

Pharmaceutical Biology, V40(7);P 494-500.

Parekh AC, Jung DH.1970.Determination of Cholesterol with ferric

acetate,Uranium Acetate and sulphuric acid-ferrous sulphate reagents , Anal

Chem;V(42);P 1523-1529