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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
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
* 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.
.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:
● Diminished adhesion
1.5.BIOCHEMICAL CHANGES
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
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:
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.
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:
Photodynamic Therapy:
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.
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)
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)
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)
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.
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)
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).
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)
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
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.
50 95 79.32 30 20.68
60
53.06
50
40.6
percentage of cell death
40
32.17
30
20.68
20
10
4.21
0
Control 50 100 250 500
μg/ml
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
Mean survival time Increase in life span(%)
Particulars
(MST) (days) (ELS)
EAC control 19±1.1
EAC control + EEPI
24±1.37 26.31
(100mg/kgbw.)
EAC control + EEPI
29±2.71* 52.63*
(200mg/kgbw.)
EAC control + 5FU
31±1.59* 63.15*
(20mg/kg bw)
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%
Group I - - -
1.8
1.6 1.5
1.4
Tumour volume (ml)
1.2 1.1
0.8 0.7
0.6 0.5
0.4
0.2
0
Group I Group II Group III Group IV Group V
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)
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.
SOLUBILITY:
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.
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.
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.
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