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7.1 Summary

Ethnopharmacology is the cross-cultural study of how people derive medicines from

plants, animals, fungi or other naturally occurring resources. Till now, the field has

focused mostly on developing drugs based on medicinal use of plants by indigenous

people.Human use of medicinal plants provides an untrapped resourse for such basic

research.For the most part, the discovery of the drugs stems from knowledge that their

extracts are used to treat one or more diseases in human. The more promising extracts are

then subjected to pharmacological and chemical tests to determine the nature of the active

components.

There are hidden treasures in these indigenous systems of medicines especially for

chronic diseases.Higher plants have been described as chemical factories that are capable

of synthesizing unlimited number of highly complex and unusal chemical substances,

whose structures could escape the imagination of synthetic chemists forever.continuous

availability of herbal medicines can only be secured through the establishment of

scientific validation.

Cancer is a multistep process that requires the accumulation of multiple genetic mutations

in a single cell that bestow features characteristic of a neoplastic cell. Cells that

undergone neoplastic transformation usually express cell surface antigens that appear to

be of normal fetal type and have other signs of apparent ―immaturity‖ and may exhibit

qualitative or quantitive chromosomal abnormalities, including various translocations and

the appearance of amplified gene sequences. Such cells display uncontrolled growth,

invasion that intrudes upon and destroys adjacent tissues, and often metastasizes, wherein

the tumor cells spread to other locations in the body via the lymphatic system or through

the bloodstream.

Cancer is a major human health problem worldwide. The management of cancer can be

done by early detection of cancer and with present local treatment methods like surgery

or radiation therapy, at least one third of patients are curable where tumor has not

metastasized by the time of treatment. In case of early micrometastasis neoplastic

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conditions local treatment is not sufficient to control the disease, indicating that systemic

approach of drug treatment is needed such can be attained by chemotherapy for effective

cancer management. At present, about 50% of patients with cancer can be cured with

chemotherapy, contributing to cure in about 17% of patients (Avjot and Rajiv 2011).

The Current conventional and modern curative methods like chemotherapy, Radiation are

ineffective or only minimally effective in malignant stages of cancer [Dodd, 2000]. In

view of the limited treatment and negative prognosis of cancer, preventive control

approaches like research for new drug molecules may ultimate cure for cancer, although

many of the currently available agents fall short of this goal. The focus of discovery and

development of new anticancer drugs through empirical screening and rational design of

new compounds increased markedly over the past few years.

A successful anticancer drug should kill or incapacitate cancer cells without causing

excessive damage to normal cells. Unfortunately, the currently available drugs in market

not meet this criterion. Developing promising candidates for clinical use with low

toxicity improves therapeutic index of novel cytotoxic drugs (Katzung, 1992).

Chemoprevention refers to the use of synthetic or natural agents to prevent, arrest, or

reverse the process of cancer development (i.e., carcinogenesis) before the occurrence of

invasion and metastasis. Chemoprevention is a promising strategy for cancer prevention

that is international in application and may be more immediate in worldwide impact than

either dietary modification or prevention of exposure to carcinogens (Greenwald, 1996).

The aim of cancer chemoprevention is to circumvent the development and progression of

precancerous cells through the use of non-cytotoxic nutrients and/ or pharmacological

agents. However, most cancer chemotherapeutic drugs affect the host normal cells.

Hence, the use of natural products has been considered a great value in the curing the

dreadful killer cells (Mehta, et al., 2011).

The discovery of new drugs effective against resistant tumors is an important and

necessary strategy in improving chemotherapy. Natural drugs have found direct medical

application as drug entities, but they also serve as chemical models or templates for the

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design, synthesis, and semisynthesis of novel substances, such as paclitaxel (Taxol),

vincristine (Oncovin) and camptothecin, in the treatment of human cancer. The currently

using well known anticancer drugs like vincristine, vinblastine and taxol are derived from

plants based on ethanopharmacological information. Attempts are being made to develop

new promising anticancer drugs from herbal sources.

In the present study four medicinal plants viz. Diospyros peregrina, Capparis Sepiaria,

Cleome viscosa, Talinum portulacifolium have been selected to evaluate the antitumor,

cytotoxic and chemopreventive effect with special reference to antioxidant activity on

various experimental animal models.

Plants were selected to evaluate their beneficiality are cited in ethnobotanical literature

for their anticancer activity.Different plant extracts were prepared and preliminary

Phytochemical studies were carried out.The preliminary Phytochemical studies of plant

extracts has shown the presence of flavonoids, triterpenoids, polyphynolic compounds

commonly.

In the present study, the methanolic extracts of plants subjected to acute toxicity studies

using mice. The results demonstated that methanolic extracts of D.peregrina, C.viscosa

and T.portulacifoilum were shown toxicity at high concentrations.The methanolic

extracts of selected plants under study were also tested for sub acute toxicity study in rats.

There is no change in organ weight, hematological parameters and biochemical

parameter. Morphological and histological characters of liver were also not altered. All

these are indication of the safety of the extracts for administration.

The present study demonstrated that oral administration of MEDP, MECS, MECV and

METP at 125, 250 and 500 mg/kg for 28 days was not toxic to wistar rats. Plant extracts

did not affect the behavior or physical examinations of the rats. There were increase in

weight gain of rats on treatement with MEDP, MECS, MECV and METP. The test drugs

did not affect hematology or clinical chemistry. Treatment-related morphological and

histopathological abnormalities were not found in liver and kidney organs/tissues

examined. These findings confirm that, long term treatment of MEDP, MECS, MECV

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and METP at sub acute doses does not lead to toxicity, so it is safe to use the methanolic

extract of selected plants for chronic in vivo studies on animal models.

The Methanolic extracts Diospyros peregrina, Capparis sepiaria, Cleome viscosa and

Talinum portulacifolium were studied for in vitro antioxidant activity by DPPH radical

scavenging method, Hydroxyl radical scavenging method and Superoxide radical

scavenging method and also against lipid peroxidantion to edivent their antioxidant

activity. The antioxidant activity exhibited various extracts was compared against a well

known antioxidant, Ascorbic acid in an attempt determine their efficacy as

antioxidants.The results were clearly indicated that the antioxidant potential of

methanolic extracts of D.peregrina, C.sepiiaria, C.viscosa and T.portulacifolium were

found to be potent free radical scavengers in in vitro model system.

The extracts also studied for cytotoxic studies by MTT assay method to prove their

cytotoxic property using human cancer cell lines MCF-7 (Breast cancer cells), HeLa

(Human cervical carcinoma), Hep G2 (Liver cancer cells). Pet. Ether extact of different

plants were not shown any cytotoxic activity against any cell lines.Methanolic and

Ethylacetate extracts were found to be cytotoxic on above stated human cancer cell

lines.MEDP has shown moderate cytotoxic activity against MCF-7 cell line, where as

EEDP has shown moderate cytoticity against HeLa when compared to HeLa and Hep-G2

cell lines.MECS was shown moderate cytotoxicity against MCF-7 and HeLa, same time

EECS shown cytotoxicity against HeLa when copared to Hep G2 Cell lines. EECV and

METP were observed with moderate cytotoxic activity against all three cell lines.EETP

and METP were shown potent cytotoxicity against MCF-7 and HeLa where as moderate

against Hep-G2. From this results it is indicating that ethlyacetate and methanolic extracts

are having cytotoxic activity. When compated to ethylacetate extract methanolic extracts

showing significant cytotoxic towards all the selected celllines, so it is confirms that

methanolic extract would be selected to screen further in vivo studies using animal

models.

From the evidences we got from our in vitro studies of cytotixicy and antioxidant

property we took up an in vivo animal experimentation to prove the antitumor,

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chemopreventive and antioxidant properties of methanolic extract of D.peregrina,

C.sepiaria, C.viscosa and T.portulacifolium.Since the plant extracts are reported to have

antitumor and antioxidant activity by ethnobotanical literature and their antioxidant

activity, toxicity profile was established by our earlier studies, we have selected classical

and traditional cancer models i.e transplantable tumor models EAC and DAL and

hepatocellular carcinoma model i.e DEN induced hepatocellular carcinoma models which

are reliable and reproducible rodent animal models and also which is capable of

mimicking clinical cancer condition in rodents to study antitumor and chemopreventive

activity with reference to antioxidant activity.We included one group of control animals

and selected 5-FU as standard drug of comparision with the test drugs as it is a common,

easily available and economic anticancer drug used in cancer treatment.

Methanolic extracts Diospyros peregrina, Capparis sepiaria, Cleome viscosa and

Talinum portulacifolium were studied for antitumor effect against two transplantable

tumors viz. Daltons ascites lymphoma and Ehrlich ascites carcinoma. Concentrations of

methanolic extracts doses 200 and 400 mg/kg were taken up for study to determine dose

dependent efficacy for 14 days of period in mice transplanted with EAC and DAL ascitic

tumor cells. After the completion of study period of 14 days, to evaluate the drugs

activity, tumor growth parameters such as life span, maximal survival time, tumor

volume, viable and non viable cell count, peretonial cell count, reduction in solid tumor

volume, hematological parameters like WBC, RBC, Heamoglobin, PCV, apart from these

liver biochemical parameters like SGOT, SGPT, Bilirubin, Albumin and total protein

levels were studied. Antioxidant property was assessed by determining the primary

oxdative stress defense enzymes viz. SOD, Catalase, GSH, GPX, GST and the status of

oxidative stress by lipidperoxidation by TBARS.

Transplantable tumor models results clearly indicated that, the antitumor effect of these

methanolic extracts of four plants is evident from the increase in lifespan, decrease in

average body weight, and increase in peritoneal cell count, reduction in solid tumor

volume and also the reversal of altered hematological parameters near to normal when

compared to the tumor control group of animals. Biochemical estimation SGOT, SGPT

and bilurubin were incresead and proten and albumin levels were decreased in tumor

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control group as compared with normal control, which are indicators of liver damage in

cancer. The results clearly indicating that upon treatment with plant extracts the altered

liver biochemical parameters comes to normal and the protective effect seen dose

dependently. Lipid peroxidation (LPO) levels were significantly increased and oxidative

tress marker enzymes SOD, Catalase, GSH, GPX and GST were decresased in tumor

control group of animals when compared to control. The altered oxidative stress

pathological changes brought back to the normal in extract treated tumor control group of

animals. Antioxidant effect of four extracts was evident with protecting against oxidative

stress with increasing free radical scavenging enzyme levels in tumor bearing animals.

The chemopreventive effect of four extracts against hepatocellular carcinoma induced by

diethyl nitrosamines was also evaluated. Chemopreventive effect of these extracts is

evident from the reversal to normal of morphological and histopathological changes, as

also biochemical parameters all which was altered by DEN. In addition, these extracts

have an antioxidant effect as reflected by decrease in LPO, and increase in SOD,

Catalase, GST, GSH and GPX enzymes which are free radical scavengers. It is likely that

chemopreventive effect is mediated through the antioxidant effect.

From the results it can be concluded that Ethyl acetate and methanolic extracts of the four

plants are selectively toxic against human cancer cell lines.Methanolic extracts also

proved to posseses free radical scavenging activity by testing usisng in vitro models..

Methanolic extracts are showing significant potent cytotoxic activity against human

cancer cell lines so they are selected for testing against transplantable tumor models and

DEN induced carcinogenesis in animal models. From the results it is observed that

methanolic plant extracts are active against Ehrlich ascites carcinoma and Daltons ascites

lymphoma amd DEN induced hepatocellular carcinoma.

All the extracts contain flavonoids. Flavonoids are known to possess antimutagenic and

antimalignacy effect (Hertog, 1992). In addition antineoplastic activity, flavonoids exert

growth inhibitory effects on several malignant tumor cell lines in vitro (Suolinna, 1975;

Markaverich, 1988; Yoshida, 1990).

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Moreover, flavonoids and Terpinoids have a chemopreventive role in cancer through

signal transduction in cell proliferation and angiogenesis (Fotis, 1997, Weber G, 1996,

Petronelli, 2009, Bishayee, 2011, Thoppil, 2011). The list of flavonoids and triterpenoids

with proven antineoplastic effect is too long one, which includes the following

1. Andrographolide, a diterpenoid lactone isolated Andrographis paniculata,

2. Bacoside A, a triterpenoid saponin isolated from Bacopa monniera.

3. Epoxy clerodane diterpene from Tinospora cordifolia

4. Lucidenic acids A, B, C and N triterpenoids from Ganoderma lucidum.

5. 25-Methoxyhispidol A, a novel triterpenoid from the fruit of Poncirus trifolata.

6. Flavonoids from Eriodictyon californicum.

7. Luteolin from edible plants.

8. Flavones from Premna odorata.

9. Eupatilin, a flavonoid from Artemisia.

10. Butenyl flavonone from Tephrosia toxicaria.

Flavonoids and Terpinoids may inhibit carcinogenesis by acting as ―blocking agents‖ by

one or more of the following possible mechanism.

1. Inhibiting the metabolic activation of the carcinogen to its reactive intermediates.

2. Inducing the enzymes involved in the detoxification of the carcinogen and

3. Binding to reactive forms of carcinogens, thereby preventing their interaction

with critical cellular targets such as DNA, RNA and proteins.

In addition, plant flavonoids could also inhibit tumor promotional events as mentioned

above. It is likely that flavonoids may emerge as a distinct group of antitumor agents.The

phytochemical studies of present study revealed that the methanolic extracts of the

selected plants under study contain flavonoids and terpenoids which may attributed for

their anticancer and antioxidant activity.

Thus observations on the methanolic extracts of D.peregrina, C.sepiaria, C.viscosa and

T.portulacifolium strongly suggest that thsese test drugs are potent antioxidants,

cytotoxic, antitumor and chemopreventive agents in treating cancer as these can play a

vital role in the modification of the disease etiology.

The biological significant role of these plants as evident from the results of present

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investigation proved the potential strength of traditional medicines in treaing cancer.

7.2 Conclusions

The following conclusions can be drawn from the results of the present investigations.

The methanolic extracts of Diospyros peregrina, Capparis sepiaria, Cleome

viscosa and Talinum portulacifolium evaluated on rats for sub acute toxicity.

There was no change in organ weight, hematological, biochemical parameters.

Morphological, Histopathological charecters of liver and kidney not altered. All

these indication of the safety of the extracts for administration.

The selected plants confirm to have potent in vitro antioxidant as evidenced by

preventing free radical scavenging activity.

The plants also confirm to have cytotoxic activity against various human cell

lines.

The preclinical pharmacological evaluation using conventional and specific model

determines antitumor and chemopreventive activity.

The methanolic extracts of D.peregrina, C.sepiaria, C.viscosa and

T.portulacifolium has antitumor and antioxidant potential which was determined

by Ehrlich ascites carcinoma and Daltons ascites lymphoma models.The

antitumor aactivity of these plant extracts were evident from increasing life span,

decrease in body weight and increase in peretonial count, recudction in tumor

volume and also reversial of altred heamotalogical, biochemical and antioxidant

parameters near to normal.

The chemopreventive effect of Methanolic extracts of D.peregrina, C.sepiaria,

C.viscosa and T.portulacifolium was determined by DEN induced hepatocellular

model. Chemopreventive ability of extracts evident from bringing back the

elevated biochemical tumor markers and oxidative stress markers to normal which

are induced by DEN.

Morphological and Histopathological changes are also normalized in drug treated

group of animals which are altered on DEN induced hepatocellular carcinoma.

The results of the study strongly recommended that the traditional medicinal

plants are important sourse of anticancer drugs. They can be used as supportive

drugs in the form of antioxidants in treating cancer.

All these extracts have shown significant anticancer and antioxidant activity.

Isolation of active constituents from crude extracts which are responsible for