01 introduction 1-4 02 aims and objective 5 03 reviwe of

III

BENEFICIAL CARDIAC ROLE OF BOERHAAVIA DIFFUSA AND

ASPARAGUS RACEMOSUS ON DOXORUBICIN INDUCED

CARDIOTOXICITY IN RATS

Ranjan Kumar Singh1*, Yogita Nirmal

2 and Mobin Ali

3

1*

Assistant Professor, Department of Pharmacology Sanjivani College of

Pharmaceutical Sciences Rajota, Khetri Jhunjhunu Rajasthan India- 333503.

2Assistant Professor, Sanjivani College of Pharmaceutical Sciences Jhunjhunu

Rajasthan- 333503.

3(M.Pharm Scholar) Arya College of Pharmacy, Jaipur, India.

TABLE OF CONTENT

S.NO CONTENT PAGE.NO.

01 INTRODUCTION 1-4

02 AIMS AND OBJECTIVE 5

03 REVIWE OF LITERATURE 06-60

04 MATERIALS AND METHODS 61-65

05 RESULT 66-75

06 DISCUSSION 76-78

07 SUMMARY AND CONCLUSION 79-80

08 REFERENCES 81-85

LIST OF ABBREVIATIONS

ALP - Alkaline Phosphate

ALT - Alanine Aminotransferase

AST - Aspartate Aminotransferase

CAT - Catalase

CNS - Central Nervous System

CHF - Congestive Heart failure

CK - Creatine Kinase

Dox - Doxorubicin

ECG - Electrocardiography

PX - Glutathione Peroxidase

GSH - Reduced Glutathione

LDH -Lactate Dehydrogenase

LPO - Lipid Peroxidase

MDA - Malondialdehyde

IV

NO - Nitric Oxide

ROS - Reactive Oxygen species

RNS - Reactive Nitrogen species

SO - Superoxide

SOD - Superoxide Dismutase

TBARS - Thiobarbituric Acid Reactive Substances

PSD- Punarnava, Satawari, With Doxorubicin.

LIST OF TABLES

S.NO TABLES PAGE. NO.

01 Experimental studies showing the use of antioxidants in the

prevention of the cardio toxic effect of doxorubicin. 33

02 Major differences between B. diffusa and Boerhaavia elegans. 44

03 LIST OF CHEMICALS USED 61

04 LIST OF DRUGS USED 61

05 MORTALITY RATE 66

06 HEART WEIGHT, BODY WEIGHT, ANDHEART / BODY

WEIGHT RATIO. 67

07 Estimation of ALP, CK, and LDH (Normal) 69

08 Estimation of ALP, CK, and LDH (DOX) 69

09 Estimation of ALP, CK, and LDH (PSD) 69

10 Estimation of ALP, CK, and LDH (PD) 69

11 Estimation of ALP, CK, and LDH (SD) 70

12 COMPARETIVE STUDY OF SERUM ENZYMES BIOMARKERS. 70

13 HEART RATE ANALYSIS 74

LIST OF FIGURE

S.NO FIGURE PAGE.NO.

1 Cardiotoxicity causing factors. 22

2 Free radical mechanism causing ROS stress 28

3 General overview of molecular mechanism and biological effects of

Anthracyclines. 31

4 Cardiotoxic and antitumor mechanisms of action of doxorubicin. 52

5 Cardio toxicity mechanisms and targets of doxorubicin. 53

6 ELECTROCARDIOGRAM. 64

7 Body weight of animal model 68

8 Heart weight of animal model 68

9 Graph of ALP of treated rats in myocardial toxicity. 70

10 Graph of CK of treated rats in myocardial toxicity 71

11 Graph of LDH of treated rats in myocardial toxicity. 71

12 NORMAL SALINE (NS) TREATED RAT. 72

13 DOXORUBICIN (DOX) TREATED RAT. 72

14 COMBINATION OD B.DIFFUSA AND ASPERGUS RACEMOSUS AND

DOXORUBICIN (PS+D) TREATED RAT. 73

15 BOARHEEBIA DFFUSA AND DOXORUBICIN (P+D) TREATED RAT. 73

16 ASPERGUS RACEMOSUS AND DOXORUBICIN (S+D) TREATED RAT. 74

17 COMPARISION OF HEART RATE OF DIFFERENT GROUP OF ANIMAL. 75

www.wjpr.net Vol 7, Issue 4, 2018. 1

Introduction

CHAPTER- 1

INTRODUCTION

Healthy human life is always cardinal for human being starting from his birth to the

end of life. The number of disease, minor level to major level, plays a key role in

disturbing the human life. Along with the modernization with the sophistication in the

life, human health faces challenges directly and indirectly from several diseases

resulting sometimes in survival and sometimes in surrender to diseases.[1]

Cancer,

remain the principal cause of death in both developed and developing countries.

Cancer known medically as malignant neoplasm is a broad group of various diseases,

all type of cancar involving unregulated cell growth. In cancer, cells divide and grow

uncontrollably and making malignant tumours, and invade nearby all parts of the

body. There are over lots of types known cancers those afflict human.

Cancer is usually treated with chemotherapy, radiation therapy and surgery. New

anticancer therapies have led to a long life expectancy for many patients; however,

treatment-related comorbities have become an issue for long-term cancer survivors.

Cardiac toxicity is one of the most feared side-effects of anticancer agents so that the

gain in life expectancy due to anticancer therapy might be countered by increased

mortality due to cardiac problems, above all heart failure, but also myocardial

ischaemia, arrhythmias, hypertension, thrombo embolism.[2]

Doxorubicin / Adriamycin (DOX) is a powerful, clinically well established and highly

efficacious anticancer agent. widely used in various neoplastic disease including

breast and oesophageal carcinomas, acute leukimias, solid tumours, kaposi’s sarcoma,

soft tissue sarcoma, Hodkin’s and non Hodkin’s lymphomas. But its clinical

usefulness is still restricted due to its specific toxicities to cardiac tissues. CHF,

Cardiac myopathy and ECG changes were demonstrated after cumulative Dox

administration.

The mechanisms proposed for cardio toxic effects of doxorubicin included

Free radical induced cardiac muscles damage.

Lipid per oxidation

Mitochondria damage

Iron-dependent oxidation damage to macromolecules


Introduction

Inhibition of cardiac coenzyme Q.o

Decrease in cardiac adenylate charge

Although the mechanism underlying the severe toxicity of DOX and other

anthracylines are not fully understood, there is evidence that the drug toxicity may be

ensure through drug free radical formation and subsequent redox cycle with O2

resulting in the generation of reactive oxygen species that SOD anion, OH- radicals

and H2O2. Tissues with less defence antioxidant defences such as heart are

particularly susceptible to injury by DOX induced oxygen radicals because it has

relatively low antioxidant enzymes such as SOD dismutase and Catalase.[3,4,5,6]

Doxorubicin (dox) is a broad-spectrum anticancer drug.[7,8]

Despite its broad

therapeutic effective-ness, the clinical use of DOX is limited by a dose-dependent and

cumulative cardio toxicity.[9,10]

Doxorubicin an anthracycline is well established and

highly efficacious drug in the fight against many kinds of cancer, small cell carcinoma

of the lung and oesophageal carcinoma[11,12]

, but its clinical use is restricted because

its specific cardio toxicity[13]

, congestive heart failure, cardiomyopathy and ECG

changes were demonstrated after cumulative doxorubicin administration.[14]

The

mechanisms proposed for cardio toxic effects of doxorubicin include free radical

induced cardiac muscles injury, peroxidation of lipid, damageing in mitochondria and

redused activity of Na+

K+

ATPase, vasoactive amine release, impairment in

myocardial adrenergic regulation, increase in serum cholesterol, triglyceride and

lower density lipoproteins. Generation of reactive oxygen species like superoxide

anion and hydrogen peroxide by doxorubicin leads to causing impairment of cell

functioning and cytolysis, because of the presence of less developed antioxidant

defence system.[15,16,17,18]

Cellular damage induced by doxorubicin is mediated by the formation of an iron-

anthracycline complex that generates free radicals, which then cause severe damage to

the plasma membrane and interfere with the cytoskeleton structure.[14]

Due to the

presence of less developed antioxidant defence mechanisms, heart is particularly

vulnerable to injury by anthracycline induced reactive oxygen species. Because

liberation of free radicals is central to the mechanism of doxorubicin induced damage

to the myocardium, considerable efforts have been made to use antioxidants and iron

chelators to protect the heart against doxorubicin toxicity.[19]


Introduction

Heart is particularly vulnerable to injury by anthracycline induced reactive oxygen

species. Liberation of free radical is central to the mechanisms of doxorubicin induced

damage to the serum enzyme like lactate dehydrogenise (LDH) and cretaine

phosohokinase (CPK). Endogenous antioxidant deficits have been suggested to play

an important role in doxorubicin induced cardiomyopathy and heart failure.[20,21,22]

Antioxidant compounds have shown protective effects in doxorubicin induced cardio

toxicity without affecting its therapeutic efficacy. There is a growing interest in the

usage of natural antioxidants as a protective strategy against the cardiovascular

problems. The antioxidant compound scavenges the free radicals produced and

protects the heart from oxidative stress.[23]

Herbal medicines are one of the important component and role of preparation of new

pharmaceutical products. A whole range of plant derived dietary supplements, plant

chemicals and pro-vitamins that assist in maintaining good health and combating

disease are now being described as functional parts. The role of medicinal plants in

disease prevention or control has been attributed to antioxidant properties of their

constituents. the main part of plant which are play important role in the protective

effect such as enzymes, proteins vitamins, caretenoides, flavonoids ,and many more

component.[24,25]

Combinations of herbal drugs, antioxidant compounds have shown protective effects

in doxorubicin induced cardiac toxicity without decresing its therapeutic efficacy.

additionally, there is a growing interest in the usage of natural antioxidants as a

protective strategy against the cardiovascular related problems in experiments such as

ischemia reperfusion and doxorubicin induced cardio toxicity.

Extracts of B. diffusa leaves have shown antioxidant and hepatoprotective properties

in pharmacological models. Punarnavine (an alkaloid isolated from of B. diffusa) has

shown some in vitro anticancer, anti-estrogenic, immune-modulators and anti-

amoebic activity (particularly against Entamoeba histolytic). Boerhavia diffusa is a

good source of antioxidants, minrals and vitamins, and may be effective against

arsenic trioxide induced cardio toxicity.[26,33]

Satavari is an important medicinal

hearbs of India. Its medicinal usage has been reported in the I.P and B.P and in

traditional systems of medicine for example; Ayurveda, Unani and Siddha. That’s

http://en.wikipedia.org/w/index.php?title=Punarnavine&action=edit&redlink=1

http://en.wikipedia.org/wiki/In_vitro

http://en.wikipedia.org/wiki/Entamoeba_histolytica

http://en.wikipedia.org/wiki/Arsenic_trioxide

http://en.wikipedia.org/wiki/Ayurveda

http://en.wikipedia.org/wiki/Unani

http://en.wikipedia.org/wiki/Siddha


Introduction

mainly known for its phytoestrogenic properties. In Ayurvedaic medicine system,

Asparagus racemosus has been shown as a rasayana, the plant has antioxidant,

immunestimulant and cardio protective effect anti-dyspepsia and antitussive

effects.[34]

According to the World Health Organization (WHO) Cancer is a leading cause of

death all over world, accounting for more than seven to eight million people’s deaths

in 2003 worldwide. In the United State, the canters for disease control and prevention

name it the second leading cause of death at more than half million people’s deaths in

2006-2007. Medical treatment continues to evolve in the fight against cancer as newer

and more effective drugs are sought with fewer side effects. Chemotherapy, is the

effective and relatively safe treatment, remains a very important part of cancer

treatment.

http://en.wikipedia.org/wiki/Phytoestrogen

http://en.wikipedia.org/wiki/Rasayana

www.wjpr.net Vol 7, Issue 4, 2018.

5

Aims and Objectives

CHAPTER- 2

AIMS AND OBJECTIVES

To find out the preventive role of combination of Boerhavia diffusa and

Asparagus racemosus against doxorubicin induced cardio toxicity in albino rats.

Observing the animals of described model for the following parameters:

1. General observation.

2. Mortality.

To evaluate the effect of combination of Boerhavia diffusa and Asparagus

racemosus on various enzyme biomarkers.

1. Estimation of LDH.

2. Estimation of CPK.

3. Estimation of ALP

To study the heart rate analysis of the experimental animals.

To study the body weight, heart weight, and heart and body weight ratio.


Review of Literature

CHAPTER- 3

REVIEW OF LITERATURE

Lipshultz SE et al.,[5]

explained the anthracycline associated cardiotoxicity in

survivors of childhood cancer that Anthracycline chemotherapy can lead to a broad

range of cardiovascular abnormalities, many of which are the progressive and of late

onset. It was concluded that with the increased success of paediatric cancer treatment,

cardiac care providers must a ssume their role in the prevention, diagnosis and

management of treatment related cardiovascular disease.

Pouna Paul et al.,[35]

developed the model of rat isolated perfused heart for the

evaluation of anthracycline cardiotoxicity and its circumvention. The anthracycline

studied were doxorubicin, pirarubicin and daunorubicin. After the all prescribed study

it was concluded that Epirubicin, pirarubicin and daunorubicin were less cardiotoxic

than doxorubicin.

Jensen RA et al.,[36]

evaluated the dose and time dependent effects of dox on the rat.

Electrocardiograph and have related the ECG alterations to cellular transmembrane

potential (TMP) changes and ultrastructural changes in preparations isolated for Dox

treated animals. It was concluded that electrophysiological changes were observed

with Dox in fact represent toxic changes.

Thippeswamy AHM et al.,[37]

investigated the effect of the aqueous extract of

phyllantus niruri against doxorubicin induced myocardial toxicity in rats. The general

observations like LDH, CPK, ALT, SOD, CAT etc were monitored after 3 weeks of

the last dose. It was concluded that the plant extract protects the myocardium from the

toxic effects of doxorubicin.

Swamy AV et al.,[38]

studied the preventive role of curcumin against dox-induced

myocardial toxicity in rats. It was concluded that the curcumin increased the level of

GSH, SOD and CAT & the biochemical and histopathological reports support the

cardioprotective effect of curcumin which could be attributed to antioxidant.

Karim S et al.,[39]

studied the protective effect of losartan, enalapril, vit. A, aspirin,

melatonin and a combination of melatonin and aspirin against doxorubicin induced

cardiotoxicity in rats. After studying the all prescribed parameters it was concluded



that the combined administration of melatonin and aspirin may offer better protections

as compared to melatonin alone against doxorubicin induced cardiotoxicity.

Shafik AN et al.,[40]

studied the role of protective agents against anthracycline

induced cardiotoxicity and nephrotoxicity by designing the animal model. After

studying all the prescribed parameters it was concluded that co administration of

either cravedilol or nebivolol was able to ameliorate up to almost contradict dox-

induced cardiac damage, glomerular filtration disturbance and renal tubular injure

with upper hand for nebivolol.

Olson DR et al.,[41]

explained that doxorubicin metabolite doxorubicinol causes

doxorubicin cardiotoxicity; that this highly toxic metabolite was produced by cardiac

tissue exposed to doxorubicin suggests that doxorubicinol could accumulate in the

heart and contribute significantly to the chronic cumulative cardiotoxicity of

doxorubicin therapy. It was concluded that the doxorubicin was more potent than

doxorubicinol in inhibiting tumour cell growth in vitro suggests that the cardiotoxicity

of doxorubicin is dissociable from its anticancer activity.

Minnoti G et al.,[42]

discussed about the double edge sword of anthracyclines;

molecular advances and pharmacologic developments in antitumour activity and

cardiotoxicity. It was also explained the recent advances that may serve as a

framework for reappraising the activity and toxicity of anthracyclines on basis of

clinical pharmacology grounds. It was concluded that anthracyclines remain

evergreen drugs with broad cinical indications but have still an improbable

therapeutic index.

Bovelli D et al.,[2]

discussed in his review about the cardiotoxicity of

chemotherapeutic agents radiotherapy related heart disease: ESMO clinical practice

guidelines. After studying all the necessary factors and parameters it was stated that;

although permanent complications tend to occur less frequently under a total dose of

40 mg, it is not a better plan to systematically limit treatment, which may be

insufficient to control the neoplastic disease.

Jaenke RS et al.,[43]

studied the six anthracyclin antibiotics with demonstrated

antitumor activity in human or experimental tumour systems. In this research



cardiotoxic potential of these compounds were compared and characterized the

myocardial pharmacokinetics in order to provide a possible explanation for

differences in cardiotoxicity. It was concluded that the degree of anthracycline

myocardial toxicity may be directly related to the relative qualitative and quantitative

accumulation of drug metabolites in the myocardium.

Bernard Y et al.,[44]

investigated the cardioprotective potential of synthetic flavagline

analogs FL1–4 in cellular and animal models of doxorubicin-induced cardiotoxicity

and explained the mechanism by which FL3 leads to cardioprotection. It was

concluded that flavaglines may protect other organs than heart against the damaging

effects of cancer chemotherapies.

Raskovic A et al.,[45]

investigated the potential cardioprotective and hepatoprotective

effects of administration of silymarin, affluent in silibinin, at a dose of 50 mg/kg

orally for a time-span of 12 days on doxorubicin induced toxicity in male Wistar rats.

According to physiological, pharmacological, microscopic and biochemical results, it

was confirmed that at the examine dose, silymarin exhibit a shielding influence on the

heart and liver tissue against toxicity induced by doxorubicin.

Rath SK et al.,[46]

explained the antioxidant activity of Rasayana herbs described in

the ayurveda. The available literature was screened and a comprehensive list of herbs

having Rasayana effect and potential anti-ageing effect was drawn. It was concluded

that the Ayurveda, the oldest documented organised system of health care has also

dealt this important health segment under aegis of Rasayana concept and also the

remaining plants should also be investigated for their anti-ageing effect.

Arola OJ et al.,[47]

studied the role of myocardial apoptosis following doxorubicin

administration; rats were exposed to 1.5, 2.5 and 5 mg/kg of. Doxorubicin and

terminated on days 1–7 in groups of five. It was concluded that the acute doxorubicin-

induced cardiotoxicity involves cardiac myocytes apoptosis, a possible preventable

form of cardiac myocytes tissue loss.

Nagy L et al.,[48]

determined a method for detection of doxorubicin-induced

cardiotoxicity by flow mediated vasodilation of the brachial artery. It was suggested

that the alterations in FMD after DOX allows for detection of patients with



insufficient antioxidant capacity and patients with insuffiecient antioxidant capacity

and patients at a higher risk of DOX-induced cardiotoxicity.

Vikrant A et al.,[6]

discussed about the various cardioprotective plants from ayurveda

and explained about 31 plants of ayurvedic origin with their various chemical

constituents and other biological activities. The review was focused on an overall

outline of plant used in ayurvedic drug for the further scientific investigation.

Koti BC et al.,[20]

determined the cardioprotective effect of lipistat against

doxorubicin induced myocardial toxicity in albino rats by doxorubicin administration

(15mg/kg for 2 weeks) and lipistat as pre-treatment (350mg/kg for 2 weeks) & then

for 2 weeks alternated with doxorubicin. It was concluded that lipid lowering and

antioxidant property of lipistat indicates the cardioprotective property against

doxorubicin induced cardiotoxicity.

Ikeda Y et al.,[50]

Reported that the androgen-androgen receptor (AR) system plays

important roles in cardiac growth and protection from angiotensin-II-induced cardio

remodelling. It was concluded that the androgen-AR system is thought to counteract

Dox-induced cardiotoxicity partly through activation of the AKt pathway and up-

regulation of Tfam to protect cardimyocytes from mitochondrial damage and

apoptosis.

Dolci a et al.,[51]

explained that the cardiotoxicity has a strong impact on patients with

cancer, in clinical and prognostic terms. Early detection was crucial for applying

preventive and supportive therapeutic strategies. The role of cardiac traponin

determination to stratify the cardiotoxicity risk was currently based on strong

evidence clearly suggesting the routine use of that biomarker.

Xin Yen-Fei et al.,[52]

explored the hypothesis that Lycium Barbarum (LB) may be

protective against DOX-induced cardiotoxicity through antioxidant-mediated

mechanisms using male SD rats. The results suggested that LB elicited a typical

cardioprotective effect on DOX-related oxidative stress and also it was concluded

that, in vitro cytotoxic study showed the antitumour activity of DOX was not

compromised by LB and it was possible that LB could be used as a useful adjunct in

combination with DOX chemotherapy.



Singh MK et al.,[53]

evaluated the combined effect of simvastin (SIM) and

hydroalcoholic seeds extract of Lagenaria Siceraria (H.L.S.S.E.) in doxorubicin

induced cardiotoxicity in Wistar rats. After performing all the necessary steps and

studying the parameters it was concluded that the pre treatment with SIM and HLSSE

may significantly reduced the DOX-induced cardiotoxicity.

CANCER

Cancer is also called malignant neoplasms in medically term. Cancer is a broad group

of different diseases, all types of cancer connecting to uncontroled cell growth. In

cancer, divide and growth of cell is uncontrollably and they form malignant tumours,

and infect nearby parts of the body. The cancer may also infected to more distant parts

of the body by the lymphatic system or by blood. Not all tumors are cancerous.

Tumor do not produce non stop, do not attack neighboring tissues and do not infect by

the body. There are over two hundred different identified cancers that badly affect

humans.[54]

Many things are known to increase the risk of cancer, use taobaccoo, certain infection,

by radiations, less physical exercise and environmental pollutants.[55]

These can

directly damage genes or combine with existing genetic faults within cells to cause

the disease.[56]

Approximately five to ten percent of cancers are entirely hereditary.

Cancer can be detected in a number of ways, including the presence of certain

symtoms and sign, diagnostic tests, or radiography and x-rays. Once a possible cancer

is detected it is diagnosed by various methods. The chances of surviving the disease

vary greatly by the type and location of the cancer and the extent of disease at the start

of treatment. While cancer can affect people any step of life. some type of cancer are

ordinary in Childs, the risk of beginning cancer normally increases with mature. In

2007, 15% people death due to cancer in all over world. Rates are rising as more

people live to an old age and as mass lifestyle changes occur in the developing

world.[57]

Cancer ills the body when injured cells divide uncontrollably to form lump or lots of

tissue called tumors. Except in the case of blood cancer where cancer prohibits normal

blood function by unusual cell distribution in the blood flow. Tumors can develop and

get in the way with the nervous system circulatory systems and digestive system and

https://en.wikipedia.org/wiki/Lymph

https://en.wikipedia.org/wiki/Cancer#cite_note-1



they can release hormones that alter body function. Tumors that stay in one spot and

show limited growth are normally measured to be benign.

More dangerous, or malignant, tumours‟ form when two things occur:

1. A cancerous cell manages to move throughout the body using the blood or

lymphatic system, destroying healthy tissue, this process is called invasion.

2. That cell manages to divide and develop, making new blood vessels to nourish

itself in a process called angiogenesis.

As tumors successfully spreads to other parts of the body and grow, invade and

destroy other well tissues, it is said to have metastasized. This route itself is called

metastasis and the result is a solemn condition that is very hard to treat.

History

Father of morden chemotherapy is Sidney farber. The first use of drugs to treat cancer

was in the early 19th century, although it was not initially intended for that reason. In

the World War I mustred gas was used for chemical warfare agent. And that was

founded to be a potent suppressor production of blood. A similar family of

compounds known as nitrogen mustred were studied at the time of world war second,

at Yale University. It was reasoned that an agent that injured the fast growing WBCs

might have a same effect on cancer. Therefore, in Dec. 1942, many patients with

advanced lymphoma (cancers of WBCs) were the drug injected by intraveinous,

relatively than by breathing the nauseating gas. This development, although

provisional, was extraordinary. At the same time, during a military operation in World

war second, following a German air raidon the Bari, several hundred people were

accidentally exposed to mustard gas, which had been transported there by air force to

prepare for possible retaliation in the event of German use of chemical war. The

survivors were later found to have very low count of WBCs.[54,59]

After world war

second was over and the reports declassified, the experience converge and led

researchers to study for other substances that might have same effects alongside

cancer. The first chemotherapy drug mustine to be develop from this line of research.

Then lots of other medicines have been discovered for cancer treatment, and the drug

development has change into a billion‟s dollar industry, even though the ethics and

restrictions of chemotherapy open by the early researchers still apply.[60]

http://en.wikipedia.org/wiki/Air_raid_on_Bari



Causes of Cancer

Cancer is ultimately the result of cells that non stop develop and not die. Normal cells

in the body go behind on systematic path of development, multidivision and selfdeath.

the Apoptosis is the process is which cell death by a preprogrammed manner, this

type of cell death is called apoptosis and when this process break down, cancer strat to

form. Distinctly normal cells, cancer cells not experience preprogrammatic death and

instead continue to divide and grow. This leads to a group of anomalous cells that

produced out of control.

Classification of cancer

There are five broad groups that are used to classify cancer.

1. Carcinomas are categorized by cells that cover up internal and external parts of

the body. for example Mouth, liver, lymphomas, lung.

2. Sarcomas are characterized by cells that cover up, fatty tissue and tissue, muscle.

3. Lymphomas are cancers that begin in the lymphatic nodes and immune system

tissues.

4. Leukaemias are cancers that begin in the bone marrow and often accumulate in

the bloodstream.

5. Adenomas are cancers that arise in theglandular tissues of the body.

Treatment of cancer

Cancer treatment depends on the type of cancer, the stage of the cancer, age of

patient, health status and additional personal characteristics.in medical science not

single methods for treatment for cancer and patients often is given a mixture of

therapies and soothing care. Treatments of cancer usually following type: surgery,

radiation, chemotherapy, immunotherapy, hormonal therapy, or genetic therapy.

Surgery

Surgery is the oldest treatment process for cancer therapy. That is possible to totally

treat a patient by surgically removing the cancer from the body. This is generally seen

in the exclusion of the testicle, breast and prostate. After the infection has increase,

however, it is almost not possible to eliminate all of the cancer cells. Surgery may

also be helpful to control symptoms such as bowel obstruction or spinal cord

compression.



Radiation

Radiation treatment, is also called radiotherapy, destroys cancer by focusing high-

frequencys rays on the cancer cells. By the effect of incidend rays, the cancer cells

damage. In the radiotherapy utilization of high-energy, gamma-rays, that are emit

from metals such as radium or high-energy x-rays that are formed in a special

machine. Untimely radiation treatments show harsh side-effects due to the energy

beams would also damage normal, healthy tissue and other part of body, but due to

technologies, improvement that beams can be more perfectly embattled. Radiotherapy

is used as a separate treatment to contract a tumor. And destroy the cancer cells these

methods also associated with leukemia and lymphoma, and it also used in grouping

with other cancer treatments.

Chemotherapy

Chemotherapy use chemicals that interfere with the process of cell division, damaging

proteins, DNA. So that cancer cells destroy. These treatments target any rapidly

dividing cells, but normal cells generally can improve from any chemical-induced

injure while cancer cells cannot. Chemotherapy is normally used to care for cancer

that has increase or metastasized because the medicines travel throughout the entire

body. It is a essential management for some forms of leukemia and lymphoma.

Chemotherapy treatment occurs in intervals so the body has time to repair between

doses. On the other hand, there are still common side effects such as hair loss,

queasiness, weakness and sickness. Grouping therapies often include multiple types of

chemotherapy and chemotherapy combined with other treatment plan.

Immunotherapy

Immunotherapy targets to get the body's immune system to fight with the tumor.

Local immunotherapy injects a treatment into an infected area, for example, to cause

swelling that causes a tumor to reduce in size. General immunotherapy treats the

entire body by administer an agent such as the protein interferon alpha that can

contract tumors. Immunotherapy is able to as well be considered distracted if it

improve cancer-fighting ability by stimulating the whole immune system and it can be

well thought-out targeted if the cure specifically tells the immune system to destroy

cancer cells. These therapies are quite new, but researchers have had success with

treatments with antibodies to the body that reduce the enlargement of breast cancer



cells. Bone marrow transplantation can also be considered immunotherapy because

the donor's immune cells will often attack the tumor or cancer cells that are present in

the host.

Hormone therapy

A number of cancers have been linked to some types of hormones, most notably

prostate and breast cancer. Hormone treatment is planned to alter hormone production

in the body so that cancer cells stop increasing or are killed completely. in Breast

cancer, hormone therapies often aimed on decresing estrogen levels (a common drug

for this is tamoxifen) and prostate cancer hormone therapies often aim on decreseing

testosterone levels. In addition, some leukemeia and lymph cases can be treated with

the cortisone hormone.

Gene therapy

The aim of gene therapy is to replace injured genes with ones that work to address a

basic reason of cancer: injure to DNA. For example, researchers are work on the

replace the damaged gene that signals cells to stop dividing (P53 gene) with a copy of

a active gene. Other gene based therapies focus on more damaging cancer cell DNA

to the point where the cell self dead. Gene therapy is a very new area and has not until

resulted in any successful treatments.

Chemo is a type of treatment that includes a drug or combination of drugs to care for

cancer. The aim of chemo is to stop and decresed the rate of growth of cancer cells.

Chemotherapy is measured asystemic treatment. That means it may affect your whole

body. Chemo drugs target fast increasing cancer cells, but they can also affect healthy

other cells that grow rapidly. The effect of these drugs on both cells often causes

toxicity. For example:

A number of blood cells that divide quickly can be injured along with cancer cells

during chemo:

o WBCs help protect the body from disease. Low WBCs is known asneutrophils. If

WBCs gets too low you could get a serious infection.

o Red blood cells carry oxygen throughout your body. Low RBCs is known as

anemiea, chest pain and more serious complications.

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o Platelets are structures in the blood that help stop bleeding. A low platelet cell

count is known as thrambocytopenia. A low platelet count can cause bruising and

bleeding.

Hairs that can be affected by chemo, leading to hair loss, also called alopecia.

Cells lining of stomach can also be affected by chemo. This can cause vomiting

and may be associated with nausea.

Chemotherapy is the treatment of cancer with one or more anti-cancer drugs

("chemotherapeutic agents"). Chemotherapy may be given with cure intent or it may

aim to long life. It is often used in conjunction with other cancer treatments. Certain

chemotherapeutic agents also have a role in the treatment of other conditions.

Traditional chemotherapeutic agent‟s act by killing cells that separate rapidly, one of

the main properties of the most cancer cells. That means chemotherapy also harms

cells that divide quickly under normal conditions: these cells present in GIT, in bone

and hair root cells. results in the most common side effects of chemotherapy:

Some newer anticancer drugs are not randomly cytotoxic, but slightly target proteins

that are abnormally expressed in cancer cells and that are necessary for their

development. These treatments are often referred to as distinct from classic

chemotherapy. And often used along established chemotherapeutic agents in

antineoplastic treatment regimen. An older and broader usage of the word

chemotherapy encompassed any chemical treatment of disease (for example,

treatment of infections with antimicrobs agents). On the other hand, this usage has

become archaicsm.

The Term Chemotherapy

The word "Chemotherapy" without a modifier usually refers to the cancer treatment,

but its chronological meaning is broader. The chemotherapy term was traditionally

used for the non-oncological references, such as the use of antibiotics (antibacterial

chemotherapy). Arsphenamine was the first modern chemotherapeutic agent and an

arsenic compound was discovered in 1909 and used to treat syphilis.[61]

This was later

followed by sulfonamides (sulfa drugs) and penicillin. There were some other uses

that have been termed as chemotherapy; such as the treatment of autoimmune diseases

such as polymyositis, multiple sclerosis, dermatomyositis, lupus and rheumatoid

arthritis.

http://en.wikipedia.org/wiki/Cancer



Principles

Cancer is the uncontrolled growth of the cells which were associated with malignant

behavior: invasion and metastasis. It is thought that the interaction between genetic

susceptibility and environmental toxins causes cancer. Amongst all chemotherapeutic

agents, most of the chemotherapeutic drugs work by impairing cell division i.e.

mitosis, efficiently targeting fast-dividing cells. They are termed as cytotoxic because

these drugs cause damage to cells. There are some drugs which cause cells to undergo

apoptosis (so-called "self-programmed cell death").

Scientists have yet to identify specific features of malignant and immune cells that

would make them uniquely targetable (accepting some recent examples, for instance

the Philadelphia chromosome as embattled by imatinib), which means that other fast-

dividing cells, for example the cells those responsible for hair growth and for

replacement of the intestinal epithelium (lining), are also often affected. Though,

some drugs have a better side effect profile, enabling the doctors to regulate the

treatment regimens to the advantage of patients in certain situations.

As chemotherapy affects cell division, tumors with high growth fractions are more

sensitive to chemotherapy (such as acute myelogenous leukemia and the destructive

lymphomas, including Hodgkin's disease), as a larger fraction of the targeted cells are

undergoes division at any time. The malignancies with slower growth rates respond to

chemotherapy much more diffidently, such as indolent lymphomas.

Drugs affect "younger" tumors (i.e., more differentiated) more efficiently, due to the

mechanisms regulating cell growth are generally still conserved. With the subsequent

generations of tumor cells, delineation is typically vanished, growth becomes less

synchronized and tumors become less approachable to most of chemotherapeutic

agents. Close to the center of some solid tumors, cell division has successfully

finished, which makes them insensible to chemotherapy. There is an additional

problem with solid tumors is that the chemotherapeutic agent often does not reach the

core of the tumour. Radiation therapy (both brachytherapy and teletherapy) and

surgery is the solution for this problem.

Over time, cancer cells become more resistant to chemotherapy treatments. Lately,

scientists have recognized small pumps on the surface of cancer cells that actively



move chemotherapy from inside the cell to the outside of the cell. Investigation on p-

glycoprotein and some others like chemotherapy efflux pumps is currently in

progress. The medications to restrain the purpose of p-glycoprotein are undergoing

testing as of 2007 to improve the efficacy of chemotherapy.

Treatment schemes

There are a number of strategies in the administration of chemotherapeutic drugs

utilised today. This treatment may be given with a restorative target or it may aim to

prolong life or to palliate indications.

Combined modality chemotherapy is the use of drugs with other cancer treatments,

for example surgery or radiation therapy and most of the cancers are now treated in

the same way. Combinational chemotherapy is a similar exercise that involves

treating a patient with a number of different drugs concurrently. The drugs vary in

their mechanism and side effects. The major benefit is minimising the chances of

resistance developing to any one agent.

In neoadjuvant chemotherapy (preoperative treatment) initial chemotherapy is

designed to shrink the primary tumor, thereby rendering local therapy (surgery or

radiotherapy) less destructive or more effective.

Adjuvant chemotherapy (postoperative treatment) can be used when there is little

confirmation of cancer present; however there is risk of reappearance. This can help

diminish chances of relapse. It is furthermore helpful in killing any cancerous cells

that have spread to other parts of body. This is frequently efficient as the newly

growing tumours are fast-dividing, and so very susceptible.

Palliative chemotherapy is given without curative objective, although simply to

decrease tumor load and increase life expectancy. For these treatments, an improved

toxicity profile is generally expected.

All chemotherapy regimens require that the patient be capable of undergoing the

treatment. Concert position is frequently used as a measure to determine either a

patient can take delivery of chemotherapy, or either dose reduction is necessitated.

Repeated doses have to be administered continuously to reduce the size of tumour

because only a fraction of the cells in a tumour dies with each treatment i.e. fractional



killing.[62]

Current chemotherapy regimens apply drug treatment in sequences, with

the regularity and extent of treatments limited by toxicity to the patient.[62]

Types

The majority of chemotherapeutic drugs can be divided into anti-metabolites,

alkylating agents, anthracyclines, topoisomerase inhibiting agents, plant alkaloids and

other anti-tumour agents.[62]

All of these drugs affect cell division or interfere with

DNA synthesis and function in some way.

Some newer agents do not directly affect the synthesis of DNA. These include

monoclonal antibodies and the new tyrosine kinase inhibitors, which openly target the

abnormalities of molecule in certain types of cancer (chronic myelogenous leukemia,

gastrointestinal stromal tumours). These are examples of targeted therapies.

In addition, some drugs that modulate tumour cell behaviour without directly

attacking those cells may be used. Hormone treatments fall into this category.[62,63]

Alkylating agents

Alkylating agents are so named because of their ability to alkylate many nucleophilic

functional groups under conditions present in the cells. Carboplatin and Cisplatin, as

well as oxaliplatin, are the alkylating agents. These agents damage cell function by

forming covalent bonds with the carboxyl, amino, phosphate and sulfhydryl, groups in

biologically important molecules.[62]

Other agents are cyclophosphamide, mechlorethamine, ifosfamide, chlorambucil,

Thio-TEPA, Carmustine, Busulfan, Decarbazine, Lomustine, etc.[62]

They work by

chemically modifying a cell's DNA.

Anti-metabolites

Anti-metabolites masquerade as purines such as mercaptopurine, azathioprine or

pyrimidines; which becomes the building-blocks of DNA. They competitively inhibit

utilization of the normal substrate or get themselves incorporated forming

dysfunctional macromolecules. They prevent these substances from becoming

incorporated into DNA during the "S" phase of the cell cycle of cell growth, stopping

normal progress and division of the cell and also they affect the synthesis of RNA.

Due to their effectiveness, these drugs are the most widely used cytostatics.



Some anti metabolites are as

Methotraxate (Folate antagonist),

Mercapturine and thioguanine, azathiorine, fludarabine (Purine antagonist),

Fluorouracil, Cytarbine (Pyrimidine antagonist).

Plant alkaloids and terpenoids

These alkaloids are derived from plants and block cell division by avoiding

microtubule function. Microtubules are fundamental for cell division and devoid of

them, cell division cannot occur. The key examples are vinca alkaloids and taxanes.

Vinca alkaloids

Vinca alkaloids bind to specific sites of tubulin, reducing the assembly of tubulin into

microtubules (M phase of the cell cycle). The chromosomes fail to move apart during

mitosis: metaphase apprehends occurs. They act in mitotic phase of cell cycle. They

are derived from the Madagascar periwinkle, Catharanthus roseus (formerly known as

Vinca rosea). These include:

Vincristine

Vinblastine

Vinorelbine

Vindesine

Podophyllotoxin

Podophyllotoxin is a plant-derived compound that is said to help with digestion as

well as used to produce the two other cytostatic drugs, teniposide and etoposide. They

avoid the cell from entering the G1 phase (the start of DNA replication) and the

replication of DNA (the S phase of cell cycle). The actual mechanism of its action is

not however known.

The substance has been primarily obtained from the American Mayapple i.e.

Podophyllum peltatum. Recently it has been investigated that an uncommon

Himalayan Mayapple i.e. Podophyllum hexandrum contains it in a much larger

amount however, as the plant is rare, its supply is limited. Studies have to be

conducted to isolate the genes involved in the production of substances, so that it

could be acquired recombinantly.



Taxanes

The prototype taxane is the natural product i.e. paclitaxel originally recognized as

Taxol and first obtained from the bark of the Pacific Yew tree. The semi-synthetic

analogue of paclitaxel is Docetaxel. Taxanes increase the stability of the

microtubules, preventing the separation of the chromosomes during anaphase.

Examples of Taxanes are as

Paclitaxel

Docetaxel

Topoisomerase inhibitors

Topoisomerases are essential enzymes that maintain the topology of the DNA.

Reduction of type I or type II topoisomerases interferes with both transcription and

replication of DNA by upsetting proper DNA super coiling.

Some type I topoisomerase inhibitors are as irinotecan, camptothecins and

topotecan.

Examples of type II inhibitors are as etoposide, amsacrine, etoposide, teniposide

and phosphate. These are the semisynthetic derivatives of the

epipodophyllotoxins, substances that are naturally occurring in the root of

American Mayapple.

Cytotoxic antibiotics

These include

actinomycin

anthracyclines

doxorubicin

daunorubicin

valrubicin

idarubicin

epirubicin, which also inhibit topoisomerase II

other cytotoxic antibiotics

Bleomycin: The Bleomycin act in a exclusive way through oxidation of a DNA-

bleomycin- Fe (II) complex and producing the free radicals, which cause damage

and chromosomal aberrations.



plicamycin

mitomycin

Anthracyclines

Anthracyclines are well established as highly efficacious antineoplastic agents for

various hemopoietic and solid tumours. A clear dose-response relation for

anthracyclines in several curative chemotherapeutic regimens has been revealed. A

decreased dose causes consequences in the inferior survival and remission rates.

However, the cardio toxicity of their agents which has been recognized for more than

20 years continues to limit their therapeutic potential and threaten the cardiac

functions of many patients with cancer.[64]

Historical perspective

Daunorubicin the frist anthracyclin antibiotic to be used and it was isolated in 1963 in

Italy from culture method of streptomyces peucetius. It is showed demonstrable

activity against a wider range of tumours in children, including soft tissue and bone

sarcomas and lymphomas, as well as lymphoblastic and myeloid leukaemia. Early

phase 1 trial involved patients with widespread disease and reports of cardiotoxicity

could only uncertainly attribute toxicity to the drug rather than the disease. With

increasing use of anthracycline, however, it becomes apparent that daunorubicin and

doxorubicin were directly cardiotoxicity in children and adult.

Epidemiology

There is wide variation in the reported frequency of both clinical subclinical

cardiotoxicity. There is a relatively low rate of early clinical cardio toxicity, since

only 1.6% of all children treated with anthracyclines have cardio toxicity effects.[65]

The prevalence of the subclinical cardiac damage has been reported to be more than

57% at a median of 6.4 years after the treatment amongst the survivors of childhood

cancers[66]

and the incidence of clinical heart failure as high as 16%, 0.9 to 4.8 years

after treatment.[67]

Distinctions in study of the population, treatment protocols and

extent of follow up could account for this wide variability. The risk of cardio toxic

effect occurring about 15 to 20 year after treatment with cumulative doses

anthracycline of 400 mg/m2 is estimated to be approximate 5% (madam). in the

normal population risk of mortality from cardiac related events is 8 times higher for

long term survivors.



Pathology in biopsy of end myocardial layer of heart with treated with

anthracyclines, its shows following symptoms

Myofibril loss

Sarcoplasmic retiuculam inflammation,

Mitochondrial inflammation,

Cytoplasmic vacuolisation,

Wide spread damage with necrosis of myocytes.

The number of myocytes present in the adult heart. Myocytes lost by necrosis are not

replaced, but the remaining myocytes increased in size to compensate and there is also

increased in the amount of interesting tissue and fibrosis. The change seen in children

and adults are the same. For children sustaining anthracyclines cardio toxicity,

myocytes loss means the left ventricular wall becomes relatively thin and particularly

fails to keep pace with pubertal or growth hormones induced growth spurts,

occasionally causing subclinical damage to become clinically overt at this time.[68]

Cardiotoxicity

Figure 3.1: Cardiotoxicity causing factors.



Cardio toxicity is a condition when damage to the heart muscle occurs. Because of

cardiotoxicity, the heart may not be capable to pump the blood throughout whole of

the body and this may be due to chemotherapeutic drugs, or other medications which

may be taking to control the disease. Cardiotoxicity, if severe or harsh, could lead to

cardiomyopathy.

Cardiomyopathy

Is often a result of treatments with chemotherapeutic medications, or may be grounds

by a group of diseases or disorders, causes heart muscles damage. Heart muscle injury

may results in a disturbance of the heart's pumping action and consequent heart

failure.

Anthracyclines are tarnished for causing cardiotoxicity. It may be caused because of

many factors, such as interference with the ryanodine receptors of the sarcoplasmic

reticulum in the heart muscle cells, because of free radical formation in the heart, or

because of development of metabolic products of the anthracycline in the heart.

Cardiotoxicity frequently presents as ECG changes (especially change in the

frequency of QRS complex) and also as arrhythmias, or as a cardiomyopathy causing

to heart failure. This caused cardiotoxicity is associated with a patient's cumulative

lifetime dose. During the treatment, patient's lifetime dose is calculated and

anthracycline treatment is frequently stopped (or at least re-evaluated by the

oncologist) upon reaching the maximum cumulative dose of the particular

anthracycline.[69]

There exist facts that the results of cardiotoxicity increases in long-term survivors,

from 2% after 2 years to 5% after 15 years.[70]

Pathophysiology of Anthracycline-induced Cardio toxicity

Cardio-toxicity is subdivided into three categories based on the time of onset: acute,

early onset chronic progressive cardio-myopathy and late-onset progressive cardio-

myopathy.[71]

Acute anthracycline-induced cardio-toxicity

Acute anthracycline-induced cardiotoxicity occurs in less than 1% of childhood

cancer patients and is defined as a severe, but temporary decrease in LV contractility

http://en.wikipedia.org/wiki/Free_radical

http://en.wikipedia.org/wiki/Electrocardiogram

http://en.wikipedia.org/wiki/Heart_failure

http://en.wikipedia.org/wiki/Anthracycline#cite_note-wow-9



seen immediately after anthracycline administration.[72]

Symptoms usually occur

within 1 week of treatment and may range from arrhythmias and electrocardiographic

abnormalities to myocarditis-pericarditis syndrome or congestive heart failure.[73]

Sinus tachycardia, possibly as a result of autonomic dysfunction, an arrhythmia, but

decrease in the QRS amplitude, causes prolongation in QTc interval and also results

in nonspecific ST segment and T-wave changes.[65]

Serum cardiac biomarker

elevations are considerably more common during this window.[77]

Discontinuing the therapy generally results in reduction in the symptoms initially,

although in many patients, particularly who received a greater cumulative

anthracycline dose, shows the permanent cardiac damage and are more likely to

develop late signs of cardiotoxicity.[65]

Early Onset of Anthracycline-induced Cardiotoxicity

Anthracycline-induced cardiotoxicity that develops during therapy or within the first

year after treatment and persists is referred to as early onset chronic progressive

cardiotoxicity and is observed in 1.6%[74]

to 2.1%[66]

of all anthracycline treated

children. In near the beginning onset of cardiotoxicity occurs, LV contractility is

reduced, most probably from anthracycline-induced damage or death of

cardiomyocytes.[65,66,71]

In addition to the positive correlation between higher cumulative anthracycline doses

and the incidence of the cardiotoxicity and the other factors are associated with a

distinctly increased risk of developing early onset anthracycline induced

cardiotoxicity. These factors include trisomy, black race and female sex, additionally

to concomitant mediastinal irradiation, or treatment with amsacrine.[65,75]

Late onset of Anthracycline induced Cardiotoxicity

Cardiotoxicity presenting at least a year after the completion of anthracycline therapy

is classified as late-onset cardiotoxicity and typically follows a chronic, progressive

course.[76,78]

Lipshultz et al., investigated that 6 years after end of anthracycline

treatment approximately 65% of survivors of childhood cancer have detectable left

ventricular abnormalities, either structural or functional.[76,77]

javascript:newshowcontent('active','references');






Late-onset anthracycline-induced cardiac failure may evolve from the inability of the

remaining cardiomyocytes to meet the demands of normal growth or other cardiac

stresses, such as pregnancy or acute viral infection.[79,80]

The resulting

cardiomyopathy may depend on the age during which the survivor was treated with

anthracyclines.[76]

Treatment before the completion of physiological growth and

development would leave fewer cardiomyocytes available for compensatory

hypertrophic remodeling of the LV.

The Clinical Presentations of Cardiotoxicity

Interestingly, although adult cancer survivors who experience cardiomyopathy as a

result of their exposure to anthracyclines generally have dilated cardiomyopathic

disease and childhood cancer survivors frequently present with a combination of both

dilated and restrictive cardiomyopathy.[72,81]

The type of cardiomyopathy that

develops in these young patients is influenced by whether they also received radiation

treatment that includes the heart. In Dilated cardiomyopathy, LV systolic dysfunction

occurs and which is more common among patients who received only anthracyclines,

but cardiomyopathy may developed from exposure to both anthracyclines and cardiac

radiation; may also occur in the later stages of patients. Initially, the patients who

taking both the cardiac radiation and also the anthracyclines present with restrictive

cardiomyopathy and therefore diastolic dysfunction, which much may afterwards

developed to systolic dysfunction.[79,82,83]

Risk Factors for Developing the Anthracycline induced Cardiotoxicity

The risk of developing clinical cardiotoxicity in both children and adults coincides

with increases in the cumulative dose of anthracycline.[81,84]

While avoiding the use of

anthracyclines in the treatment of certain cancers would be the most adequate strategy

to prevent cardiotoxicity[85]

this may negatively influence tumor response and

ultimately the survivals. The most severe cardiac discrepancy associated with

anthracycline toxicity occurs at the highest cumulative doses. Though, lower doses

are not totally safe either, as the symptoms of cardiotoxicity occurs even been

reported in patients receiving only low doses of anthracyclines.[78]

This variability in

susceptibility may be partially explained by genetic polymorphisms of certain genes,

although a few numbers of studies have discoverd this possibility and have reported

conflicting results.[84,86,87,88]





One study found that children with a cumulative anthracycline dose greater than 550

mg/m2 were more than five-times as likely to occurrence of cardiotoxicity, than the

children who received low cumulative doses[65]

van Dalen et al. reported that the

estimated incidence of developing anthracycline-induced clinical heart failure

increased with both time since treatment and cumulative anthracycline dose (5.5% at

20 years after the start of anthracycline therapy if treated with a cumulative dose less

than 300 mg/m2 and 9.8% if treated with a cumulative dose exceeding 300 mg/m

2),

over a mean of 8.5 years (median: 7.1 years; range: 0.01–28.4 years).[89]

The

incidence of anthracycline-induced clinical heart failure in this cohort was 2.5% and a

cumulative anthracycline dose of 300 mg/m2 or more was the only independent risk

factor (relative risk [RR]: 8.0). The authors concluded that 10% of children receiving

a cumulative anthracycline dose of 300 mg/m2 or more will eventually experience

anthracycline-induced clinical heart failure.

Lipshultz et al. reported that a higher cumulative anthracycline dose was associated

with worsened LV structure and function with regard to both afterload and

contractility.[76]

More precisely, several studies have noted that cardiac dysfunction

becomes more noticeable in patients who received a cumulative anthracycline dose

that exceeds approximately 250 mg/m2.[90,91]

Additionally, a higher dose rate,

previously treatment with anthracyclines, longer action, younger age at diagnosis and

radiation therapy involving the heart have all been identified as risk factors for

developing late cardiotoxicity. Another risk factor for developing anthracycline-

induced clinical heart failure in children is being female. For the girls, this risk is

approximately increased by 4 times as great as it is for male childhood cancer

survivors treated with anthracyclines.[92]

Lipshultz et al. reported that LV contractility

of female childhood cancer survivors 8 years after completing doxorubicin treatment

was significantly worse than that of their male counterparts. A few explanations have

been posited for this difference. The Doxorubicin clearance is lower in those patients

with higher BMI, which usually associates with increases in the percentage of body

fat.[74]

Girls, on average, have a larger body-fat: lean-mass ratio than the males, which

possibly will increase the amount of anthracyclines and stored in adipose tissue,

consequently exposing nonadipose tissues to higher concentrations of anthracyclines

for a longer period of time.[75,93]






It is important to note that not all of the presented risk factors are identified by all of

the studies that considered them. Though, differences in the study proposed and

sample size between these studies may partially explain the different results. For

instance, some studies only observed the high-risk subgroup of the survivors, or more

aggressive form of the cancer, a priori. These children are liable to take delivery of

higher doses of doxorubicin in order to treat the cancer more aggressively, while the

standard risk children do not get such high doses.[75,76,78]

Mechanisms of the Anthracycline induced Cardiotoxicity

Despite more than three decades of the use, it is yet unclear actually that how

anthracyclines exert their chemotherapeutic activity and induce the cardiotoxic

changes. Antineoplastic activities of the doxorubicin can be derived from the

inhibition of topoisomerase II, free-radical generation, activation of signalling

pathways, DNA intercalation and binding and apoptosis. Similarly, causes of

anthracycline-induced cardiotoxicity appear to be multifactorial. A significant

component may cause production of free radicals and the presence of the redox

related damage, which happen through both the enzymatic and the nonenzymatic

pathways and result in the accumulation of iron. Anthracycline-generated free radicals

induce lipid peroxidation, which ultimately produces membrane damage. In addition,

mitochondrial damage, increased Ca2+

current along with inhibition of sarcoplasmic

reticulum function and decreased activity of Na, K-ATPase, have all been implicated

in doxorubicin-induced cardiotoxicity. The down regulation of myocardial TNF-α and

doxorubicin binding to bivalent cations (Ca2+

, Mg2+

, Cu2+

and Zn2+

) may also be

contributively.

Mechanism of the Anthracycline toxicity in Cardiomyocyte

Anthracyclines enter cardiomyocytes by passive diffusion and stimulate the

production of free radicals, which leads to cell damage. The Anthracyclines also

directly and indirectly inhibit the gene transcription; also inhibit the mitochondrial

functioning and also energy production within the cell. Compared with other organs,

the heart is especially susceptible to the anthracycline induced damage, in part, due to

anthracyclines high affinity for the cardiolipin. Cardiolipin is a unique mitochondrial

phospholipids involved in various stages of mitochondrial membrane dynamics and

the mitochondrial apoptotic process. During the initial stages of apoptosis, apparently




in synchronization with death receptor stimulation and the generation of reactive

oxygen species (ROS) cardiolipin can become peroxidised. Peroxidation of

cardiolipin can interfere with the localization of heme iron of cytochrome C and

results in its release and also the release of additional apoptogenic factors, from

mitochondria. While it appears that peroxidation of cardiolipin plays a critical role in

releasing cytochrome C from the inner part of mitochondrial membrane, the

cytochrome C itself be able to catalyze cardiolipin peroxidation. Cardiolipin's cellular

role is multifaceted and further investigation into its contribution to apoptosis is

warranted.

Anthracycline exposure activates both ROS-dependent and ROS-independent

pathways that may ultimately lead to a sizeable loss of cardiomyocytes through

necrosis and apoptosis. Other types of cell death, including senescence and the

autophagy, may also put in to the anthracycline induced cardiomyopathy. Myofibrillar

loss and cytoplasmic vacuolization, can be caused by the dilation of the sarcoplasmic

reticulum in the myocardial cells, these are the most common chronological findings

in patients with anthracycline-induced cardiomyopathy. The degree of cellular

damage corresponds with the cumulative anthracycline dose administered. The

primary compensatory mechanism for anthracycline-induced cardiomyocyte loss is

the cardiomyocyte hypertrophy, which finally results in a series of physiological

changes that lead to the development of the cardiomyopathy. These types of changes

include the decrease in left ventricular (LV) contractility, the thinning of the LV wall,

increase in LV afterload, fibrosis all of which are cause to reduced LV function.[24]

Figure 3.2: Free radical mechanism causing ROS stress.



In addition, the heart's relative lack of strong antioxidant defence mechanisms and

high rate of oxidative metabolism make it particularly vulnerable to damage by iron

and free radicals, which consecutively, can increase the permeability of the cell

membrane. Substantial experimental data supports the involvement of iron in

anthracycline-induced cardiotoxicity, which includes the ability of an iron chelator

and dexrazoxane, to alleviate anthracycline-induced cardiotoxicity. In this

investigation of Cascales et al. was investigated that whether anthracyclines increases

the iron concentration in the heart and if it done then whether the HFE genotype

helping to regulate iron deposition. They retrospectively examined the cardiac iron

concentrations, HFE genotype and the cardiac events in 97 consecutive necroscopies

from adults with hematological and the solid neoplasms. From these, about 48

patients had been treated with anthracyclines and the other 49 patients had been

received either some other type of the chemotherapy (24 patients) or some

nonchemotherapeutic form of therapy (25 patients) and were used as the controls.

When the tests compared with the control group, the cumulative doxorubicin dose

greater than 200 mg/m2 was associated with increased cardiac concentrations of iron

(490 vs 240 µg/g; p = 0.01), regardless of the patient's transfusion history or level of

iron into the liver. Some mutated haplotypes were associated with greater iron

deposition (282C/63D, p = 0.049; 282Y/63H, p = 0.03) and the haplotype C282Y-

Y/H63D-H actually increased cardiac iron accumulation through its interactions with

anthracyclines. The investigators from all studies stated that the HFE helps regulate

iron accumulation after exposure to anthracyclines and that this iron accumulation is

independent of a patient's systemic iron load.

Other possible contributors to anthracycline-induced cardiotoxicity include reduced

expression of mRNA encoding for the sarcoplasmic reticulum Ca2+

-ATPase, which

results in diminished cardiac contractility and transcriptional changes in intracellular

cardiomyocyte ATP production. In addition, prolonged exposure to anthracyclines

may also lead to DNA damage resulting from depressed cardiac glutathione

peroxidase (GSHPx) activity and respiratory chain defects. Respiratory chain defects

are associated with the production of the free radicals and also which can continue to

produce even after the anthracycline treatment, ultimately causal to late anthracycline

induced cardiomyopathy. This production of radicals may also release cytochrome C

from mitochondria, ultimately leading to apoptosis.[9]




Focusing on liver and cardiac tissue, in his study Doroshow et al. determined the

alterations in concentrations of catalase, superoxide dismutase and GSHPx (all

enzymes noted for their ability to detoxify activated oxygen) in response to treatment

with doxorubicin administered through intraperitoneal injection. Experiments in

murine models suggested that the GSHPx in cardiac tissue was the selenium

dependent. The superoxidase and Catalase concentrations in the cardiac tissue were

<0.6% and about 27% of that found in the liver tissue, correspondingly, but the

GSHPx concentrations were similar in the both types of tissues. Though, after the 6

weeks of selenium reduction, the GSHPx activity was decreased to less than 20% of

the baseline and in this selenium depleted condition, marked doxorubicin induced

toxicity found in animals receiving only 15 mg/kg intraperitoneally. Distinctly,

neither the the level of cardiac superoxide dismutase, nor the concentrations of

hepatic superoxide dismutase or GSHPx were affected at the same dose. From all the

studies the authors stated that the selenium dependent GSHPx, collectively with

superoxide dismutase, to a great extent helps detoxify ROS in the cardiac tissues and

that the doxorubicin or diet induced reduction of selenium would impair the heart's

ability to efficiently dispose of lipid peroxides and hydrogen peroxide.

Although redox-induced damage is likely involved in anthracycline-induced

cardiotoxicity, a number of verification proposed that it is not only the mechanism.

Numerous studies of a variety of redox inhibitors, or the ROS scavengers, given

concurrently with anthracycline treatments have found no statistically significant

levels of the cardioprotection. The incapability of these agents to improve

anthracycline-generated cardiotoxic damage does not substantiate redox-induced

damage as a stand-alone cause of anthracycline-induced cardiotoxicity.[107]

Pointon et al. compared the cardio-toxicity induced by doxorubicin with that induced

by 2,3-dimethoxy-1,4-naphtoquinone (DMNQ) in murine models to investigate the

adequacy of the redox hypothesis of anthraxcycline-induced cardio-toxicity.[107]

DMNQ has limited ability to stop DNA replication or gene transcription but it is a

superior redox cycling agent than doxorubicin (-183 mV compared with doxorubicin's

redox potential of -328 mV). The investigators postulated that if redox damage was

the primary cause of anthracycline induced cardiotoxicity, then the DMNQ should

cause considerably more cardiac damage than the doxorubicin. Acute results to





DMNQ or doxorubicin introduction proposed that anthracycline-induced

cardiotoxicity is not primarily a result of general redox stress, however somewhat

from the reduction of the electron transport chain in the mitochondria. In his study,

Pointon et al. stated similar responses in a chronic revelation murine model, although

they did not contain the supporting data.

General overview

Figure 3.3: General overview of molecular mechanism and biological effects of

Anthracyclines.[52]

From the analysis of above figure, it seems that some of these interactions are directly

responsible for the mitochondrial dysfunctions, other are consequence of a general

modification of these drugs in a cellular system. As shown in the fig. 3.3 overview in

mainly four factors are responsible for the mitochondrial dysfunction.

a. Anthracycline determine a release of some mitochondrial enzyme, probable

through membrane disruption. The release of cytochrome cis the beginning of a

cascade that‟s lead to the cell apoptosis.

b. The high affinity of anthracyclines to lipids leads to disorder to itochondrial

membrane and to an indirect inhibition of lipid bound enzymes.

c. A direct inhibition of mitochondrial enzymes could be demonstrated only in the

case of cytochrome c oxidase.

d. Production of free radicals, in particular in mitochondria enzyme systems, lead to

a huge number of cells injure, as calcium release, as lipid peroxidation and

oxidative stress in general. This ability can be enhanced in the presence of metal

ion able to form reactive complex with the Anthracyclines.



Strategies to reduce the cardio toxicity of Anthracyclines[12]

In the treatment of chemotherapy, anthracyclines are use over 40 year and depict

intensive research efforts; no significant progress has been made in improving their

cardiac safety. It is presumed that the postulated cardio toxic mechanisms in

anthracyclines are not the same effects as those operating in tomour toxicity. This

allows hopes that it might be possible to achieve cardio protection without loss of

antineoplastic efficacy.

Effort in this direction as so far focused on

a. Reducing the total cumulative doses.

b. Co therapy with protective agents.

c. Changing the chemical structure of the anthracyclines moiety.

d. Encapsulation of anthracycline in liposomes.

e. Development of new safe derivative.

Reducing the total cumulative doses

Chronic cardiotoxicity and heart failure may any time competition of doses of

anthracyclines drugs and occurs more frequently in patients given cumulative doses of

doxorubicin 550mg/m2.[12]

A lower doses limit would reduce the incidence of serious

cardiotoxicity, but these strategies would deny treatment with a potent and effective

agent to many who would tolerate much higher doses and potentially benefits from its

maximum antineoplastic therapeutic effects and the present approach is to administer

the Dox up to point beyond which further therapy would result in cardiotoxicity. This

required an ability to monitor for cardio toxicity and the safety the cumulative doses

of doxorubicin.[22]

By this approach the significant reduction in the incidence severe

doxorubicin cardio toxicity has been achieved.

Co therapy with protective agents

An anthracyclines have very potent anti-tumour effects and their extensive use will

persist until effective and safer alternative have been identified. An extensive array of

compounds has been evaluative in various experimental models for their potential to

reduced dox cardio toxicity. On the basis of a perceived ability to modulated some of

the biochemical alternations that accompany dox administration. Most research in this

area has focused on antioxidant agent or iron chelotors. Antioxidant enzymes such as



SOD or Catalases, Vitamin E, lycopene, sulfer containing antioxidents are shown to

protect against the cardio toxicity of anthracyclines in several experimental modals.

Table 3.1: Experimental studies showing the use of antioxidants in the

prevention of the cardio toxic effect of doxorubicin.

ANTIOXIDANT MODEL RESULT

Vit.E Rat

Abolition of ST segment

evaluation, reduction of cardiac

enzymes. (CPK, LDH)[23]

Vit. E and N-acetyl

cysteine Rat

Protection to word lipid tissue

cardiac and hepatic paroxidation.

Protection of action potentials

and cardiac activity.[24]

N-(2-mercapto-

propionylglycine) Rat

Protection towards lipid tissue

cardiac peroxidation[25]

Lyopene Rat

Reduction of cardiac tissue lipid

peroxidetion and cardiac enzyme

release.[26]

Gluthione Rat

Protection towards hepatic and

cardiac lipid tissue peroxidation.

And cardiac enzymes release.

Reduction of cardiac histological

ultreation.[27,28]

Sod,catalase Rat

Prevention of dox-induced

reduction of cardiac myocytes

contractility.[29]

Changing in the chemical structure of the anthracyclines moiety[67]

Anthracyclines determine a release of some mitochondrial enzymes, propbly through

the membrane distruction. The discharge of cytochrome C is the commencement

(starting) of a cascade that‟s lead to cell apoptosis. Slightly structural change can

determine a completely changed reactivity of these drugs with enzymes or metal ions

and therefore change the ability to produced free redicals. These changes can also lead

to change affinity constant to certain and lipophlic characters.

The encapsulation of anthracyclines in liposomes

The Liposomal encapsulated anthracyclines are used in preclinical studies and result

in a different pharmacokinetics profiles. pegylated liposome‟s used for clinical

applications(caetly, doxil) resulted in longer circulation times and a higher anti-tumor

activity with respective to the free drug. The changed pharmacokinitics and

distribution of liposomal encapsulated anthracyclines allow rising the administrative



doses in chemotherapy without increasing the cardio toxicity. The resulting higher

cumulative drug concentrations, led to an increase of other dose dependent side

effects, that‟s less dramatic with respect to the cardiomyopathy but also therapy

restraining. There is one of the main serious problems during the anthracycline

chemotherapy is PPE. These ulcer active cutaneous lesions on hand and feet strongly

through to be related to a free radical induced damage of endothelial cells and

keratinocytes. It is important to note this severe side effect occurs also during the

administration of the free drug and is not related to the concomitant presence of the

liposome. The knowledge of the affinity of the chemotherapies to certain lipids, i.e.

cardiolipin, in the preparation formulation of the liposomes could be exploited in

order to avoid the intreaction with mitochondrial membrane. The ability to generate

free radicals scavengers inside the liposome.

Development of new safe derivatives

Many attempts have been made to be finding new anthracyclines with lower toxicity

than daunorubicin and doxorubicin. Thousands of analogous have been synthesised

and tested, but few have found their way into clinical use. Among these so called

second generation anthracyclines are epribucin and idarrubucin.

Epribucin is the semi synthetic derivative of doxorubicin with a similar mechanisms

and scope of efficacy to its mother component. epirubicin can be given at a higher

cumulative doses. Its shows no reduction in toxicity, compare to doxorubicin, when

given therapeutically equal doses. Despite an improvement in therupatic index, the

cardiac risk remains. The daunorubicin derivative idrabucine has a wide spectrum of

efficacy and can give orally. However, dispute some promising finding any reabile

improvement in cardiac safety is in dought.[131]

PLANT PROFILE

Introduction

It is been recorded in history that herbal Medicine have been used as form of

treatment for the pain. The exploration of the chemical contents from herbs,

pharmacological action and phyto-chemical screening would provide the basis for

producing the new lead molecules in strategic favour of herbal product drug

discovery. The objective of researchers is the discovery and development of isolating

a new less toxic, active and efficient molecule for systemic activities. The biologically



active agents from natural sources have always been of great interest to working on

various diseases. Since hundred yeras ago tribal community are using these traditional

knowledge system to medicate various type of diseases. They use different parts of

plant as a source of drug through trial and error method and the process is experienced

over hundreds of years, which says that the medicinal plants have been in the focus as

life saving drugs right from the beginning of the human life. The medicinal plants

have been the object of research in both systematic and advanced areas of plant

sciences. The traditional knowledge of these herbal recipes is popular among the

indigenous and local communities. Even present scenario the Tribal communities are

solely dependent on herbal medicines for their medication. So they are using herbs

against different. They have preserved the wealth of traditional knowledge as a part of

their belief and customs. They are practicing these methods, generation after

generation, successfully. Apart from medicinal uses phytochemical components

which are environment friendly, economical and effectively shows anti corrosive

properties and also phyto-compounds are used as biofuels.

Plant 1

Boerhaavia Diffusa

Boerhaavia Diffusa Linn. (Nyctaginaceae), common name is punarnava. In the Indian

system of medicine, is a permanent steal herb found throughout the waste land of

India. The roots are reputed to be diuretic and laxative and are given for the treatment

of various diseases. The Boerhaavia Diffusa has ancient medicinal use in different

societies from the times of the B.C. The herbal medicine has evolved and changed

through the years. A number of plant products have been identified through phyto-

chemistry and the extract of their different plant parts are useful in many diseases

without any side effects.

Geographical Distribution and Habitat

Boerhaavia diffusa, consisting of more than 40 species is distributed in tropical, sub-

tropical regions and warm climate. It‟s found in, America, Australia and Malay

Peninsula, Sudan extending to China and Islands of the Pacific. Among 40 species of

Boerhaavia, 6 species are found in India, namely B. diffusa, B. erecta, B. rependa, B.

chinensis, B.Hirsute and B. rubicund.



Boerhaavia diffusa in India is found in warm regions of the country and throughout up

to 2,100 m altitude in the Himalayan region. It is a permanent, distribution hogweed,

commonly occurring abundantly in misuse places, ditches and marshy places for the

period of rains. The plant is also cultivated to some extent in West Bengal.

Pharmacognosy

Scientific Name: Boerhaavia diffusa Linn. Syn.; B. repens vare. Diffusa

Family: Nyctaginaceae

Family Name: Hogweed, Horse Purslane Common Indian Names

Gujarati: Dholia-saturdo, Moto-satoda.

Hindi: Snathikari

Canarese: Kommegida

Marathi: Tambadivasu

Sanskrit: Punarnava, Raktakanda

Bengali: Punurnava

Tamil: Mukaratee-Kirei

Telugu: Punernava

Useful Parts: Root leaves and seeds.

Phytochemistry

The Boerhaavia diffusa plant contains a large amount of following compounds. For

example, Steroids, Alkaloids, flavonoids, lignins lipids, triterpenoids, carbohydrates,

proteins and glycoproteins. Punarnavine C17H22N2O. Melting point: 236–237°C.[43,44]

Boeravinone A-F. hypoxanthine 9-Larabino furanoside, ursolic acid, punarnavoside,

lirodendrin and a glycoprotein having a molecular weight of 16– 20 kDa have been

isolatedand studied in detail for their biological activity. Punarnava also contains

arachidic acid, α-2-sitoesterol, palmitic acid, tetracosanoic, stearic, urosil acid,

Hentriacontane, β -Ecdysone, triacontnol etc. Phytochemical screening of the roots

from garden-grown in vivo plants of B. Diffusa of different ages shown that the

highest alkaloid content 2% accumulate in the roots of 3-yearold mature plants. The

herb and roots are wealthy in proteins and fats. The herbal contains 17 amino acids,

with 7 essential amino acids and the root contains 15 amino acids, including 6

essential amino acids. Plant contained large amount of potassium nitrate, as well

punarnavine also. Previous researchs show the presence of flavonoid, alkaloid,



steroid, triterpenoid, lipid, lignin, carbohydrate, proteins and glycoprotein in B.

Diffusa.[45,55]

Other Chemical Constituents

Many rotenoids have been isolated from the roots of the Boerhaavia diffusa. Plant

also includes a series Pharmacological Potential of Boerhaavia diffusa boeravinones

viz., boeravinone A, B, C, D, E, F. Punarnavoside, a phenol glycoside, is reportedly

present in roots. C-methyl flavone also has been isolated from Boerhaavia diffusa

roots. Two known lignans viz., liriodendrin and syringaresinol mono-β-D-glycoside

have been isolated. Presence of a purine nucleoside hypoxanthine 9-L-arabinose,

dihydroisofuroxanthone-borhavine, phytosterols, have been isolated from the plant. It

contains about 0.04% of alkaloids known as punarnavine and punernavoside, an anti-

fibrinolytic agent. It also contains about 6% of potassium nitrate, an oily substance

and ursolic acid. The seeds of this plant contain fatty acids and allantoin and the roots

contain alkaloids. The green stalk of the plant has also been reported to contain

boerhavin and boerhaavic acid.

Medicinal Uses

According to Ayurveda, Punarnava is in biter test, astringent to bowels, leucorrhoea,

anaemia, inflammations, heart illness, asthma. The leaves are useful in indigestion,

cancer, spleen swelling and abdominal pains. According to Unani medicine, the

leaves are appetizer, alexiteric, useful in opthalmic, in joint pains. Seeds are tonic

expectorant, carminative, useful in backache, scabies. The seeds are considered as

promising blood purifier.

Boerhavia diffusa is believed to get better and guard eyesight. B. diffusa has diuretic

properties and is used by diabetics to decresed blood sugar. Boerhavia diffusa has

shown antibacterial activity, mainly against G- bectriea. Extracts of B.diffusa leaves

have demonstrate antioxidant and hepatoprotective properties in pharmacological

models. Punarnavine has shown some in vitro anticancer, anti-estrogenic, immune-

modulatory and anti-amoebic activity B. diffusa is a source of anti-oxidants and may

be efficient against arsenic tri oxide (an effective drug used against acute

promyelocytic leukemia) induced cardio toxicity.

http://en.wikipedia.org/w/index.php?title=Punarnavine&action=edit&redlink=1

http://en.wikipedia.org/wiki/In_vitro



Pharmacological and biological activity

The plant has gained lot of importance in the field of phytochemistry because of its

various pharmacological and biological activities such as immune-modulatory effects,

immunosuppressive activity, anti-metastatic activity, antioxidant activity, anti diabetic

activity anti proliferative and anti-estrogenic activity, analgesic and anti-inflammatory

activity, antibacterial activity, antistress and adoptogenic activity,

antilymphoproliferative activity, nitric oxide scavenging activity, hepatoprotective

activity, anti-viral activity, bronchial asthma, anti fibrinolytic activity,

chemopreventive action, genetic diversity analysis, anticonvulsant activity.

Antitumor Activity

Cancer chemo-preventive effect of B. diffusa was evaluated on 7 12-dimethyl

benz(a)anthracen (DMBA) induced skin papillomagenesis in male Swiss albino mice

(6-7 weeks old). The cancer chemo-preventive efficacy was assessed by its ability to

modulate the actions of enzymes allied with drug metabolism and biefficient

modulators reduced the availability of ultimate carcinogen metabolites in the

epithelial phase. the important enlargement in the activities of hepatic phase I & II

system. enzymes and antioxidant enzymes, glutathione peroxidase, glutathione

reductase, superoxide dismutase, catalase and glutathione level were observed when

rat were fed by oral gavage with B. diffusa extract at a dose level of 125 mg and 250

mg/kg body weight for a period of 15 days in laboratory. This lead to an assumption

that the inhibition of tumorigenesis by the plant extract might have been executed

either by preventing the formation of active carcinogens from their precursors or by

augmenting detoxification process, prevent promotional trial in the mouse skin

through free radical scavenging mechanism.

Antioxidant Activity

The evaluation of the antioxidant potential of ethanolic extract of Andrographis

echioides and B. diffusa was carry out by determining the levels of enzymatic and

non-enzymatic antioxidants. The results showed that both the plant extracts possessed

significant levels of enzymatic and non-enzymatic antioxidants. The results of the

enzymatic and non-enzymatic antioxidants in Andrographis echioides and Boerhavia

diffusa exhibits that they possess preventive and productive role to maintain the cell

survival, cellular interaction and maintenance of cell membrane architecture.



Andrographis echioides and B.diffusa have effective and therapeutic antioxidant

potential against various inflammatory diseases 61. The study was undertaken to

evaluate antioxidant activity of Chloroform, Ethanol and Ethyl acetate fraction of

Boerhaavia Diffusa L roots which might have improved its hepatoprotective action.

Invitro nitric oxide search activity, the percentage inhibition was 71.36%, 33.75%,

23.88%. in the extracts of ethanol, chloroform and ethyl acetate at 250mg/ml. when

compared with Curcumin at 62 mg/ml showed only 84.7% inhibition respectively.

The ethanol extract and ethyl acetate extrats showed a biphasic response whereas the

chloroform extract showed a dose dependent relative increase. In DPPH radial

scavenging activity, the ethanol extract showed 81.94% inhibition and the chloroform

extract showed 42.58% inhibition at 1000mcg/ml compared with 88.02% inhibition

by Quercetin. The present results propose that roots of B.diffusa were found to make

known antioxidant potential which supports the use of this plant in traditional

medicine.

Cytological activity

The extract of B. diffusa exhibited a strong depressive effect on the mitosis of C.

Jagus roots. The study was conducted using B. diffusa extract, the mitotic index of the

control experiment was found to be 5.27. There was a negative correlation between

the concentrations of the treatment extracts and the mitotic indices obtained from their

action. This points to an inhibition of mitosis by this extract. Inhibition of the mitotic

index increased significantly with an increase in the concentration of treatment

solution of B.diffusa. This again shows a very negative correlation between the

concentration of the extract and the mitotic indices produced by the observed action.

Owing to the ability of the root extracts of B. diffusa toaccumulate metaphase and

hence inhibit mitosis, it is possible to use these extracts as an alternative to the rather

expensive colchicine for cytological studies.

Bronchial Asthma

Dried leaves of Punernevacan be used in dhoomapana in treatment of bronchial

asthma. The leaf decoction is said to be an excellent expectorant when decocted with

punarnava (Boerhaavia diffusa) and then combined with ginger juice and black

pepper.



Anti Fibrinolytic Activity

A study evaluated the effect of anti-fibrinolytic agents; α-aminocaproic acid (α-ACA),

tranexamic acid (AMCA); anti-inflammatory drugs (indomethacin, ibuprofen,

naproxen); and root extract of B. diffusa on endometrial histology of IUD-fitted

menstruation of monkeys. It is effective in reducing edema, swelling and in increase

the amount of deposition of fibrin and platelets in the vessel lumen.

Anti-Convulsant Activity

The study was carrired out to investigate the alcoholic root extract of B.diffusa and its

various fractions as well as liriodendrin fraction for exploring the possible role of

liriodendrin in its anti-convulsant action. Air dried roots of B. diffusa was extracted

with methanol by cold maceration process. The methanol soluble fraction of extract

thus obtained was successively extracted to obtain liriodendrin fraction and two side

fraction, that is, chloroform division and phenolic compound division. Anti-

convulsant activity of methanolic extract and its different division, that is liriodendrin

division and phenolic compound division were studied in pentylenetetrazol-induced

seizure. The crude methanolic extract of B.diffusa and its liriodendrin division

showed a dose-dependent protection against PTZ-induced convulsions. The

liriodendrin fraction showed important protection in opposition to seizures induced by

BAY k-8544. These findings reiterate the anti-convulsant action of methanolic extract

of B. diffusa roots and also it can be concluded that the observed anticonvulsant

activity was due to its calcium channel opposit action, as this action was reserved only

in the liodendrin division, which has additionally been complete by important anti-

convulsant activity of liriodendrin division in BAY k-8644-induced seizures55. That

Study showed the crude methanolic extract of B. diffusa and its liriodendrin fraction

showed a dose-dependent protection against PTZ-induced convulsion.

Anti-proliferative and Anti-estrogenic Activity

Antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia

diffusa (BME) in MCF-7 breast cancer cell lines. Boerhaavia diffusa extracts

exhibited a strong inhibitory effect on the proliferation of human breast cancer cells in

vitro and the antiestrogenic effects are mediated by ER. Phytochemical studies have

revealed the presence of alkaloids, flavonoids, phenols and saponins in BME. The



antiestrogenic activity shown by the extract may be attributed to these diverse

compounds.

Antidiabetic Activity

The study indicates that Boerhaavia diffusa and ethanolic extracts exhibit significant

anti-hyperglycemic activities in alloxan induced as well as streptazotocin induced

dieabitic rats. They can also recover the condition of diabetes as indicate by

parameters like body weight with serum cholesterol and triglyceride levels. The

number of functionally intact β-cells in the islet of langerhance organ is of decisive

importance for the development route and result of diabetes. The renewal of β-cells in

diabetes has been studied in several animals. The all β-cell mass reproduce the

balance in between the renewal and loss of these cells. It was also suggested that

renewal of islet β-cells following destruction by alloxan may be the primary cause of

the recovery of alloxan-injected guinea pigs from the effects of the drug. In alloxan-

induced diabetes, Epicatechin and Vinca rosea extracts has also been shown to act by

β- cells renewal. Comparable effects in streptozotocin treated diabetic animals were

details report by pancreas tonic, sylvestre leaf extracts. In the present studies, injure of

pancreas in streptazotocin treated diabetic control rats and regeneration of β-cells by

glibenclamide was observed. The comparable renewal was also shown by methanolic

extracts of Boerhavia diffusa. A study was carried out to investigate the effects of

daily oral administration of aqueous solution of Boerhaavia diffusa L. leaf extract

(BLEt) (200 mg/kg) for 4 weeks on blood glucose concentration and hepatic enzymes

in normal and alloxan induced diabetic rats. A imperative decrease in blood glucose

and significant increase in plasma insulin levels were observed in normal and diabetic

rats treated with BLEt42. Chloroform extract of B. diffusa leaf produced dose-

dependent reduction in blood glucose in streptozotocin-induced NIDDM rats

comparable to that of glibenclamide. The results indicate that the decrease in blood

glucose produced by the extract is probably through renewal of pancreatic beta-cells

action.

Antibacterial Activity

A effective anti-bacterial activity against gram+ and gram

- bacteria publicized by the

leaves of B.diffusa may be due to the plants chemicals present in the leaves. Ethanol

extract show inhibitory a cause on gram + bacteria, and all gram

-- bacteria selected for



the study. Methanol extract showed inhibitory effect against all gram+ bacteria

selected for the present study except M. luteus and gram-negative bacteria. The anti-

bacterial activity of the different extracts of the stem bark of Prosopis cineraria (Linn.)

Druce was estimate by the agar on form diffusion method. The aqueous and ethanolic

extracts of B.diffusa leaves had action on E. coli, S. Aureus and P. Aeruginosa. This

activity happen at changing concentration, representing that the plant extracts limited

active principle with broad antibacterial spectrum. E.coli show the highest

receptiveness in ethanolic extract, go after by S.aureus and the least at risk was P.

Aeruginosa. In aqueous extract P. aeruginosa explain the chief resistance, followed by

S. aureus and E.coli exhibited the least susceptibility. The antimicrobial action of the

different extracts increased with increase in concentration. The results of the

investigations support the ethnomedicinal use of this plant by local practitioners.

Results from that study showed that the aqueous and ethanolic extracts of B. diffusa

had antibacterial action on E. coli and P. Aeruginosa.

Antistress / Adaptogenic / Immunomodulatory Activity

Hydroethanolic extract (70%) of B.diffusa and an herbal formulation PHF-09 contain

B. Diffusa was compared for their stress free action using cold self-control strain

model. Stress was inducing by subjecting to cold restraint. Due to cold restraint

pressure there was an difference in the intensity of biochemical factor like glucose,

triglycerides, cholesterol, which were by regularize subsequent the administration of

HEBD and PHF-09. HEBD and PHF-09 were found to have similar anti-stress action.

The ethanol extract of roots of B. diffusa was estimate for antistress, adoptogenic

action in mice, by spin endurance test and cold restrains stress and the extract showed

superior load patience in immunomodulatory action was revealed by increased carbon

relase, indicating stimulus of the reticuloendothelial method. There was an raise in

DTH response to SRBC in mice, matching to cell referee immunity and representative

stimulatory effects on lymphocytes and accessory cell types.

Adaptogens is useful in both adrenal hyperstress as well as adrenal hypo tiredness. By

definition, an adaptogen involve the means for bidirectional or regularize property.

The most important adaptogens for the adrenals consist of Panax and siberian

Ginseng, withaniea sominfera, B. diffusa and tulsil Leaf Extract. B.diffusa has the

capability to carry both adrenal over and under activation. In stressful situation it has



established the capability to barrier the elevation of serum cortisol and prevents the

inhibition of the immune system that takes place with eminent cortisol. Then again, B.

diffusa has also demonstrated the ability to improve cortisol levels with end stage

adrenal exhaustion.

Hepatoprotective Activity

The hepatoprotective activity of roots of different diameters were collected in three

seasons, rainy, summer and winter and examined in thioacetamide in poisonous rats.

The results showed that an aqueous extract (2 ml/kg) of roots of diameter 1-3 cm,

collected in the month of May (summer), exhibited marked protection of a majority of

serum parameters, LDH,CK,ALP thereby suggesting the proper size and time of

collection of Bd L. Roots for the most attractive results. Further, the studies as well

prove that the aqueous form of drug (2 ml/kg) administration has more

hepatoprotective activity than the powder form; this is probably due to the better

absorption of the liquid form through the intestinal tract. The hepatic protective

activity of Boerhaavia diffusa L. roots showed marked protection of serum parameters

in thioacetamide toxicity in animals. besides, the aqueous extract of thin roots

collected in the summer has more activity suggesting the proper time and type of root

collection for the most desirable result. The investigation also validates the use of B.

diffusa L. roots in hepatic ailments by the several tribes in India.[180]

An alcoholic

extract of whole plant Boerhaavia diffusa given orally exhibited hepatoprotective

activity against experimentally induced carbon tetrachloride hepatotoxicity in rats.

The extract as well formed an enhance in normal bile flow in rats suggesting a strong

choleretic activity. The extracts do not explain any signs of toxicity up to an oral dose

of 2 g/kg in mice.

Analgesic / Anti-Inflammatory Activity

The Decoction (DE) or Juice (JE) of the leaves of Boerhaavia diffusa were used to

study the antinociceptive effect in chemical (acetic acid) and thermal (hot Plate)

models of hyperalgesia in Mice. The DE, raised the pain thresholds during the first

period (30 min) of observation. In the acetic acid-induced abdominal writhing in rat,

pretreatment of the animals by naloxone (6 mg/kg.) considerably reversed the

analgesic effect of morphine and JE but not that of DE. The experiment shows that the

lively antinociceptive standard of B. diffusa is mainly in the juice of leaves and has a



significant antinociceptive effect when assessed in these pain models.[182]

Ethanol

extract of leaves at dose of 400mg/kg exhibitedmaximum anti-inflammatory effect

with 30.4, 32.2, 33.9 and 32% with serotonin, carrageenin, dextran, histamine and

induce rat paw edema experimental, respectively. Ethanol extract of stem bark also

exhibited COX-1 and IC50 value of 100 mg/ml proving the drug use in the treatment

of inflammatory condition. Anti-inflammatory activity was assessed using extract of

latex of plant by using a carragenan induced inflammatory mode.

Anti-inflammatory activity

Ethanol extract of leaves at dose of 400mg/kg exhibited maximum anti-inflammatory

effect with and 32% with carrageenin, serotonin, histamine and dextran are effect on

rat paw oedema experiment method, respectively. Ethanol extract of stem bark also

exhibited COX-1 and IC50 value of 100ng/ml proving the drug use in the treatment of

inflammatory condition. Anti-inflammatory activity was assessed using extract of

latex of plant by using a carragenan induced inflammatory model.

Table 3.2.: Major differences between B. diffusa and Boerhaavia elegans.

Characters Boerhaavia diffusa Boerhaavia elegans

Plant A perennial herb from a fusiform root An erect glabrous shrub

Stem

Prostrate, decumbent or ascending, 4-

10mlong,rather slender, divaricately

branched

Annual, woody below,

glabrous above, thinly

pubescent close to the support.

Leaves

Opposite, two of a node unequal,

broadly ovate or orbicular, obtuse to

rounded or cordate at the support.

Or blong-lanceolate, obtuse

and often mucronulate at the

top

Flowers

In pendunculate, glomerulate clusters

arranged in slender, long stalkcked,

axillary or terminal corymbs

In large, lax, much branched,

leafless, glabrous compound

cymes above the leaves

Fruit

Obovoid or sub-ellipsoid, curved

over, a little cuneate, under, broadly

and bluntly 4-ribbed, awfully

glandular throughout

Flowering and Fruiting Throughout the year in Indian

conditions

September to December in

Indian conditions

PLANT 2

Asparagus racemosus

In recent years shatavari herb has become a popular ayurvadic medicine in all over

world. Women in the West have been using it ever since studies showed that it

contains phyto-estrogens hormones, the precursors of estrogen hormones. Its finding



made it popular all over world. Shatavari aretwo types which are widely used,

Asparagus racemosus wild that called shatavari, and the vegetable, Asparagas

officinalis linn.

Shatavari is widely use in Ayurvedaic medicine system. According to the Ayurveda

texts like the Charak Samhita, Susruta Samhita, and Astanga Samgraha, shows its

ancient use as early as Vedic Time. Shatavari is highly appreciated in Ayurvedaic

formuation, due to its potential not only menopausal health, but health in all stages of

a female‟s life cycle. Ayurvedic record literally claims that shatavari strengthen a

female to the point where she is capable, of producing, thousands of healthy ovam.

Long before current studies were published, the great commentator “Pandit Hem Raj

Sharma”, In the Kashyap Sahita”, it‟s clearly says that shatavari effective in female

health. In particular, noted the advantage of shatavari as a galactagogue. As such, it‟s

also affecting the infant‟s health indirectly. Shatavari suitably exemplifies a central

principal of Ayurveda; the use of this herbal drug as both, medicine and food.

Asparagus racemosus (Shatavari) is recommended in Ayurvedic texts for the

prevention and treatment of ulcers, gastric problems and galactogogue. A. racemosus

has also been used successfully by some Ayurvedic practitioners for nervous

disorders. Shatawari has different names in the different Indian languages. In Nepal it

iis called kurilo.the name shatawari means “curer of hundred diseases” (shat:

hundred”; vari:” curer”).

HABITAT

This climber growing in low jungles is found all over India; especially in Northern

India. The plant is a climber growing to 1-2m in length found all over India.

DESCRIPTION

Asparagus racemosus is plant with a woody stem that sends runners out, has pinpoint

like leaves with white flowers, Scandant, much branched spinous under shrub with

tuberou, small root, stock bearing numerous fusi form tuberous roots 30-100cm thick

leaves reduced to minute chaffy scales & spines. Cladodes acicular 2-6 hate, falcate

finely acuminate flower white, berries 7mm in diameter, globose, 1-seeded, red

(Sharma et al., 2000). The Himalayas up to an altitude of 1500 m.



Leaves, flowers and fruits

Satavar has small needle like phylloclades (photosynthetic branches) that are equal

and shine green. In July, it produces minute, white flowers on stem, prickly stems,

and September it fruiting time, producing blackish-purple, globular berry.

Roots

It has an adventitious root system with tuberous roots that measure about 2m in

length, tapering at both sides, with bunch in plant.

Pharmacological activity

Asparagus racemosus has been shown to mitigate the discomfort due to Amlapitta

(Acid dyspepsia with or without ulcer) on 109 cases in a clinical Study at CRIA

DEHLI.

Antibacterial activity

Anti secretory and antiulcer activity

Antidepressant activity of Asparagus racemosus in rodent models.

Anti-inflammatory effects

Anti-cancer

Antioxidant

Antitissuve

Enhances memory and protects against amnesia in rodent models.

Antilithiatic

Hepatoprotective effects in mice fed a high-fat diet.

Aphrodisiac.

Diuretic

Teratogenic

Immune modulators (extract), Immune adjuvant,

Oestrogenic effect

Neuroprotective

Anti-Stress

http://en.wikipedia.org/wiki/Flower

http://en.wikipedia.org/wiki/Adventitious

http://en.wikipedia.org/w/index.php?title=Amlapitta&action=edit&redlink=1



Chemical constituents

Asparagamine A

Alkaloid with antitumor activity, against a variety of cell lines was isolated from the

dried roots and subsequently synthesized to allow for the construction of analogs.

Saponins: Shatavaroside A and shatavaroside B, mutually with a saponin

filiasparoside C, it was isolated from the roots of Asparagus racemosus.

5 steroidal saponins, shatavarins6-10, with 5 more, shatavarin I, shatavarin IV,

shatavarin V, immunomodulator have been collect by the roots of satahvari.

Medicinal Uses

Asparagus racemosus is an important medicinal plant of tropical India. Its medicinal

usage has been statement in the Indian in traditional systems of medicine such as

Ayurveda, Unani and Siddha. It is mainly known for its plant estrogenic properties. In

Ayurveda, A. racemosus has been described as a rasayana herb and has been used

extensively as an adaptogen to increase the non-specific resistance of organisms

against a variety of stresses. As well use in the treatment of motion problem, the plant

also has antioxidant, antidyspepsia and anti-tussive effects. Plants for a in future can‟t

take any responsibility for any adverse effects from the use of plants. Always ask for

advice from a trained sooner than using a plant medicinally. Antispasmodic,

Aphrodisiac, Demulcent, Digestive, Diuretic, Galactogogue (to improve breast milk),

Infertility, Women‟scomplaints. Root is use in Diarrhea as well as in cases of chronic

colic and dysentery. The root was boiled with some bland oil and it is used in various

skin diseases „root is boiled in milk and the milk is administered to Shatavari (this is

an Indian world meaning a woman who has a hundred husbands) is the most

important herb in ayurvedic medicine for dealing with problems connected women‟s

fertility. It is taken internally in the treatment of loss of libido, infertility, menopausal

problem, threatened miscarriage, stomach ulcer, hyperacidity and bronchial infection.

Externally, it is used to treat the stiffness in the joints. The fresh root is taken for the

treatment of dysentery. The whole satavari plant is utilized in the treatment of brain

complaints, diabetes, diarrhea and rheumatism. It is also Used in Management of

behavioral disorder and minimal brain dysfunction. The rhizome is a soothing tonic

that acts mainly on the digestive, circulatory, respiratory, female reproductive organs

etc, antispasmodic, aphrodisiac, demulcent, diuretic, galactogogue and refrigerants.



DRUG PROFILE

Doxorubicin Hydrochloride

Discovery of doxorubicin an antitumor antibiotic, in the early 1960 represented a

major advancement fight against cancer. As the drug found to be very effective in a

verity of solid and soft human malginancy, however, the cardio toxicity is well

recognizes side effect of doxorubicin (anthracycline). that limit the total amount of

drug administrated and can cause heart failure in some patient. Early retrospective

study from the 1970sdemonstrted the heart failure and doxorubicin are related. with

the incidence of complication increasing sharply when the cumulative dose increased

550mg/m2. of the body surface. The incident of heart failure was 4% when the

cumulative dose of dox in between 500 to 550mg/m2. but its increased 18% when dox

is 551mg/m2 to 600mg/m2.and to as much as 36% when the total dose was at least

601%mg/m.

The ECG changes associated with doxorubicin cardio toxicity include various

arrhythmias, most commonly sinus trachcardiya, flatting of t waves, prolongation of

qt intervals and loss of r wave voltage. The incidence of these changes has been

reported to be 11% to 29%.

Myocardial damage as seen on myocardial biopsy, increase liner with increasing

cumulative dose of dox. but clinically the incidence of cardio toxicity is more

apparent at cumulative dose greater then 400-450mg/m2. This study is also made is

clear that CHF was a dose dependent phenomena. an incidence of >5%. was seen at a

500-550mg/m2 body surface are, increasing to >30%. at over 601%mg/m

2. Thus, the

dose of 500mg was considered the upper limit in order to minimize the risk of dox

cardio toxicity and CHF. Subsequent studies showed that cardio toxicity could occurs

at a much smaller dose of doxorubicin in patients with risk factor such as age, some

underlying heart condition, pervious and concurrent radiation therapy and

concomitant chemotherapy.

Doxorubicin has still remained in use because of its high antitumor activity. It also

forms a valuable component of various regimen of chemotherapy. There is still a need

to find ways to minimize the cardio toxic effects.



Description[193]

Doxorubicin (doxorubicin hydrochloride.C27H29NO11. HCL) cytotoxic anthracycline

antibiotic isolated from the culture of streptomyces paucities ver. caesius. In the

chemical structure of doxorubicin we found a naphthacenequinonone nucleus linked

through a glycosides bond at ring atom to amino sugar, daunosamine.

Chemical description: chemically, doxorubicin hydrochloride is: 5, 12-

napthoacenedione, 10-[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-,8,9,10-

tetrahydroxy-8-(hydroxyacetyl)-1-methoxy, hydrochloride(8S-cis). Mol. weight of

doxorubicin: 579.99.

Doxorubicin binds to nucleic acids and specific interaction of the planar anthracycline

nucleus with the DNA double helix. The saturated end of the ring contains abundant

hydroxyl groups adjacent to the amino sugar; the anthracycline ring is lipophilic in

nature. Producing a hydrophilic canter. The molecule of doxorubicin is amphoteric in

nature, containing acid in the phenol ring groups and a basic purpose in the sugar

containing amino group. Its binds to cell membranes and plasma proteins.

Doxorubicin hydrochloride (Doxorex, doxorubicin hydrochloride for injection. USP)

is a sterile, red-orange in colour, lyophilized powered for intravenous or in the case of

local regional treatment of tumours by slow intra-arterial infusion. Doxorubicin

hydrochloride for injection is available in 10mg, 20mg and 50 mg single dose vials.



Each 10, 20 and 50 mg of Doxorubicin HCL, USP. And its presentation id each vial

contains 10 mg of doxorubicin hydrochloride as a freeze-dried powder, to be

dissolved in 5ml of water for injection (WFI) and its packaged in either single dose

polypropylene vials. And preservative-free, ready to use solution. Each millilitre of

solution contains 2 mg of doxorubicin HCL. Inactive ingredients sodium chloride

0.9%. I.P.

Biological activity

The mode of action of doxorubicin is connected to the ability of the antibiotic, to bind

to DNA and inhibit the synthesis of nuclide acid. The cell culture studies have

verified swiftly cell penetration by the antibiotic and main localization is in the

perinucleolar chromatin rapid inhibition of mitotic activity and nucleic acid synthesis

have also been demonstrated together with the appearance of chromosomal

aberrations.

Animal study on doxorubicin has shown that the cytotoxic agent is active in a

spectrum of experimental tumour and is immunosuppressive. However it does give

rice to a verity of toxic effects such as cardiac toxicity in rats.

Mechanisms of action of doxorubicin

The mechanisms of action of doxorubicin induced cardiac toxicity remains unknown,

but it has become possible to distinguish between the mechanisms underlying

doxorubicin antitumor effect and the mechanisms responsible for cardiac toxicity. The

proposed mechanisms for dox induced injury are cellular damage mediated by

reactive oxygen and nitrogen species. The discordance is probably associated with

different mechanisms mediating the acute and chronic cardio toxicity. In fact, a

number of studies using antioxidant supplements have failed to show a relevant

protective effect against chronic cardio toxicity. Therefore, cardiac toxicity may also

be related to some unknown non- ROS mechanisms. For example a decrease in the

mitochondrial calcium release channel and fatty acid metabolism in the myocardium.

The exact causal mechanisms of doxorubicin induced cardio myopathy remain

unclear, but most of the evidence indicates that free radicals are involved. The

chemical structure of doxorubicin is level to the generation of free radical and the

oxidative strain that results connect with cellular injury. Increases oxidative stress

may lead to a variety of sub cellular change in the myocardium, including the slow



loss of myofibrils and vacuolization of myocardial cells, changes typical of

doxorubicin induced cardio myopathy. And administration of doxorubicin is

associated with a decrease in the presence of the endogenous antioxidants and an

increases in free radicals, that‟s by oxidative stress also increased, which is followed

by the development of cardio myopathy and heart failure.

DOXORUBICIN MECHANISM

Antitumor action and cardiac toxicity

The sub cellular basis for doxorubicin induced cardiomyopathy as well as its

antitumor action was earlier thought to involve a common pathway. Though, more

recently it has become possible to differentiate the mechanisms underline these dual

effect of doxorubicin.

1. Antitumor action

Free radical injury might be a mechanism of doxorubicin anti tumour action because

doxorubicin administration has been shown to produce free radicals. Another in point

supporting the concept of free radicals-mediated anti-tumour action is that

doxorubicin forms an adarimycin-iron complex by binding with iron. This binding

can also form a complex with DNA and these complexes can stimulate the production

of oxygen (partially reduced forms). Because of free radical production in

neighbourhood of DNA strands, DNA damage as well as breaking of strands occurs.

In various literatures it was revealed that relatively high in vitro concentration of dox

was used to demonstrate the free radicals-medicated DNA damage in tumour cells.

The dox use in vivo condition were much lower, suggested that a free radicals

mechanisms might not be the primary cause of the antitumor activity of doxorubicin.

This point was supported by a multitude of studies in which the addition of different

anti-oxidants dose not compromises doxorubicin cytotoxicity in a variety of tumour

cells.

There were the other mechanisms which explain the inhibition of DNA replication by

dox and thus its cyto toxicity and anti tumour action, without involvement of free

radicals.



A direct intercalation of dox between DNA base pairs interferes with DNA

replication; that was a cooperative interaction between dox and DNA. Dox mediated

inhibition of DNA topoisomerase II was also shown to inhibit the DNA replication.

Adriamycin topoisomerase II – DNA complex prevents repairing of the broken DNA

strands.[201]

Antitumor action of doxorubicin at low concentrations, independent of free radical

mediation was reconciled with the free radical mediated cardio-toxic effect of the

drug seen at higher cumulative doses in a scheme shown in figure 3.4.

Fig. no. 3.4: Cardiotoxic and antitumor mechanisms of action of doxorubicin.[203]

Molecular mechanisms

Existing evidence points to a complex situation with a multitude of molecular

mechanisms involved in doxorubicin induced impairment of cardiac energetic and

other cellular targets, finally lead to cardiac dysfunction.



Fig. 3.5: Cardio toxicity mechanisms and targets of doxorubicin.

An important factor, which can mediate toxic action of doxorubicin, especially in

mitochondria, was high affinity binding of dox to cardiolipin, anionic Phospholipids

in eukaryotic energy metabolism. cardiolipin with its particular ability to interact

more or less specifically with many protein was very important not only for

mitochondrial structure and purpose, but also for in general cardiac energy

metabolism as well as for cell survival. 88 87. binding of doxorubicin would modify

membrane properties and changes the Phospholipids‟ environment and function of

numerous crucial mitochondrial integral membrane proteins which depends upon

cardiolipin in their function.

Cardiolipin bounded doxorubicin would also induced dissociation of cardiolipin

associated peripheral proteins from the inner mitochondrial membrane, e.g.

Cytochrom C and CK. That would affect electron transport chain and energy

channelling as well as a favour of initiation of program cell death. Toxicity of

mitochondrial, mostly cardiolipin bounded dox was mediated by oxidative stress,



which was represented a particular threat to cellular energetic in the myocardium and

was considered as the name mediator of doxorubicin cardio toxic action.

Heart tissue is rich in mitochondriathats by they heavily relies on oxidative

metabolism and thus produced significant amount of free radicals. Accumulation of

redox active adriamycin in these organelles would enhance mitochondrial production

of free radicals of oxygen and as more recently evidenced also of nitrogen species.

Quinine present in Dox compound can be converted in to the semiquinone form by

the reaction of one electron reduction. The Dox semiquinone can consequently

transfer an electron to the oxygen molecule (O2) to form superoxide anion radicals

(O2-).

[203] The latter can dismutateto form hydrogen peroxide (H2O2) and further

hydroxyl radicals or may react with nitric oxide to form peroxynitrite (ONOO-). The

noxious action of these reactive compounds could explain phenomena such as:

The triggering of apoptosis in mitochondrial cells.

Membrane damage (peroxidation of lipids)

Effects on genetic expression.

Changes in protein activity.

Damage to DNA.

The particular susceptibility of the myocardium relative to other tissue rests on its

relative lack of enzymes such as dismutase and catalases, which are responsible for

the breakdown of reactive oxygen species. The heart is particularly protected against

the oxidative stress.

Per oxidation of membrane phospholipids associated with decrease in the membrane

fluidity and also oxidation or nitration of proteins has been reported after Dox

treatment. Oxidative and nitrostative stress interfere with many aspects of cardiac

purpose, inducing between others energetic inequality, mitochondrial permeability

transition and apoptosis and activation of various related signalling pathways. In

particular the response of cardiac Ca2+

to DOX can be closely related with the

perturbation of heart energy homeostasis. Dox induced alterations in Ca2+

ion

handling and their possible consequence are already reported.



It is to note, that the actual extent and impact of oxidative and nitrostative stress

produced by Dox in different toxicity models and in patients, are a matter of

discussion. Interestingly, quinine-containing molecules can exert their effect

independently of free radicals by directly affecting some sensible protein residue, e.g.

reactive sulfahydryl groups and because of this group Dox can interact with the

cardiac gene expression as they contains sulfahydryl groups, in particular by down

regulating genes of several enzymes implicated in energy metabolism.

Even though such interference with DNA is considered important for the anticancer

action of the drug and it may also affect macromolecular biosynthesis of the heart.

Additionally, oxidative stress has been implicated in Dox-impaired gene expressions.

Fragmentations and proteolysis of Dox modified proteins were shown to occur in Dox

treated mitochondrial preparations. And also, to lower biosynthetic rate and enhanced

degradation rate, compact cellular levels of soluble proteins and metabolites in

doxorubicin challenged cells can occurs through leakage. This is well known for

cytosolic, CK, the plasma level of which serve as clinical markers of different cardiac

pathologies, including Dox toxicity.

On the other hand, it should be noted that certain gene and coding proteins implicated

in energy metabolism have been found unregulated in a mice model of chronic

Doxorubicin cardiac toxicity. This may imitate induction of a protective or

compensatory reply. additional mechanism some part to the toxic effect of

doxorubicin in cardiac tissue can be mediated by iron or Dox metabolites and by an

important damage of endothelial origin.

Communally, the response of cardiac energetic to DOX involves a complex crosstalk

between different pathways and mechanisms.

Clinical pharmacology

Pharmacokinetics study has shown that intravenous administration of labelled

doxorubicin is followed by a rapid fall in the plasma levels, accompanied however by

slow urinary and binary excreta. this is probably due to binding of the antibiotic to

tissues. the initial distribution have life of approximately 5.0 min suggests rapid tissue

uptake of doxorubicin, while its slow elimination form tissue from tissue is reflected



by a terminal half life of 20 to 4 hour. Steady-state distribution volumes exceed 20 to

30 l/kg and are indicate of extensive drug uptake into tissues. Urinary excretion as

determined by flourimetric methods accounts for approximately 5% of the

administration dose in 5 days; biliary excretion is the major elimination route, with

40-50% of the administered dose recovered in the bile or faeces. In seven days:

impaired liver function causes slower excretion of the drug and in consequence

increased accumulation in plasma and tissues dox dose not cross the blood brain

barrier.

Pharmacokinetics

The Pharmacokinetic study, determined in patients with various type of tumours

undergoing either single or multi-agent therapy have shown that doxorubicin follows

a multiphase disposition after intravenous injection.

Distribution: The primary division half-life of approximately 5 minutes suggests

rapid tissue uptake of doxorubicin, while its slow elimination from tissue reflected by

a terminal half-life of 20 to 48 hours.

Binding of doxorubicin and its major metabolite, doxorubicin, to plasma proteins is

about 74 to 76% and is independent of plasma concentration of doxorubicin up to

1.1g/ml. Doxorubicin was detectable in the milk up to 72 hour after therapy with

70mg/m2 of doxorubicin given as a 15-minute intravenous infusion and 100mg/m

2 of

cisplatin as a 26 hour i.v. mixture. The peak absorption of doxorubicinol in milk at 24

hour was 0.12g/ml and AUC up to 24 hours was 9.0g/ml while the AUG for

doxorubicin was 5.4g/ml. doxorubicin dose not the blood brain barrier.

Metabolism

Enzymatic reduction at the seventh position and cleavage of the daunsamine sugar

yields aglycone which is accompanied by free radical arrangement, the general

making of which may contribute to the cardio toxic activity of doxorubicin.

Disposition of doxorubicinol in patients is formation rate limited, with the terminal

half-life of doxorubicinol being similar to doxorubicin. The relative exposure of

doxorubicinol, i.e., the ratio between the AUG of doxorubicinol and the AUG of

doxorubicin, compared between the doxorubicin range of 0.3 to 0.7.



Execration

Plasma clearance is in the range 324 to 809 ml/min/m2 and is predominately by

metabolism and biliary excretion. approximately 40% of the dose appears in the bile

in 5 days, while only 5 to 12% of the drug and its metabolites appear in the urine

during the systemic clearance of doxorubicin is significantly reduced in obese women

with ideal body weight greater than 130%.there was a significant reduction in

clearance without any change in volume of distribution in patients when compared

with normal patients with less than 115% ideal body weight.

Contraindication

Patients should not be treated with doxorubicin if they have any of the following

conditions, baseline neutrophil count <1550 cells/mm3.; major liver injury; recent MI,

severe myocardial lack, major arrhythmias, earlier treatment with complete

cumulative doses of doxorubicin and other anthracycline or hypersensitivity to

doxorubicin, any of its excipients, or other anthracycline or anthracenediones.

INDICATIONS AND USAGE

Doxorubicin hydrochloride Injection, USP and Doxorubicin HCl for Injection, USP

have been used successfully to produce regression in disseminated neoplastic

conditions such as acute lymphoblastic leukaemia, acute myeloblastic leukaemia,

Wilms tumour, neuroblastoma, soft tissue and bone sarcomas, breast carcinoma,

ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, gastric

carcinoma, Hodgkin‟s disease, malignant lymphoma and bronchogenic carcinoma in

which the small cell histological type is the most responsive compared to other cell

types.

Doxorubicin is as well show for use as a component of adjuvant therapy in women

with evidence of axillaries lymph node involvement following resection of primary

breast cancer.

Drug interactions

Doxorubicin is extensively metabolized by the liver. Change in hepatic function

induced by concomitant therapies may affected doxorubicin metabolism,

pharmacokinetic, therapeutic and GIT actions, may be increased when doxorubicin is

used in combination with other cytotoxic drugs.



Drug Intreaction

Dabigatran etexilate

P-Glycoprotein inducers i.e. doxorubicin can

reduce the serum concentration of dabigatran

etexilate.

Digoxin The anti neoplastic agent decreased the effect

of digoxin.

Telithromycin Telithromycin may reduce clearance of

Doxorubicin.

Terbinafine Terbinafine may reduce the metabolism of

Doxorubicin.

Cyclosporine

Cyclosporine results in increases in AUG for

both doxorubicin and doxorubicinol possibly

decreases in clearance of parent drug.

Progesterone

Progesterone was given intravenously to

patients with advanced malignancies at high

doses .its enhanced doxorubicin-induced

neutropenia and thrombocytopenia was

observed.

Verapamil

A learn of the effects of verapamil on the acute

toxicity of dox in mice relevative higher initial

peak concentrations of dox in the heart with a

higher incidence and severity of degenerative

changes in cardiac tissue resulting in a shorter

survival.

Phenobarbiton Increcses the elimination of doxorubicin.

Streptozocin That inhibit hepatic metabolism of doxorubicin.

Cytarabine

Necrotizing colitis manifested by typhlitis,

bloody stool. and severe and some time fetal

infection have been associated with a

combination of doxorubicin given by i.v.

ADVERSE REACTIONS

Dose limiting toxicities of therapy are myelosuppression and cardiotoxicity. There are

other reactions which were reported as:

Cardiac

Cardiotoxicity is a known risk of treatment with anthracycline. Anthracycline induced

cardiotoxicity can be apparented by early (or acute) or late events. Early cardiotoxity

includes mainly sinus tachycardia and/or electrocardiogram (ECG) abnormalities such

as non-specific changes in ST-T wave. Tachyarrhythmia includes premature

ventricular contractions and bradycardia, ventricular tachycardia and also

atrioventricular and bundle-branch block have also been described. These type of

effects generally does‟nt predict consequent development of delayed cardiotoxicity,

http://www.drugbank.ca/drugs/DB06695





are infrequently of clinical importance, and are usually not well thought-out a

suggestion for the suspension of doxorubicin treatment.

Delayed cardiotoxicity usually develops late in the course of therapy with doxorubicin

or within 2 to 3 months after the termination of treatment, but in later events, it takes

several months to years after completion of treatment. Later cardiomyopathy is

manifested by a reduction in LVEF and/or signs and symptoms of congestive heart

failure (CHF) such as tachycardia, pulmonary edema, cardiomegaly and

hepatomegaly, oliguria, pleural effusion, etc and also subacute effects i.e.

pericarditis/myocarditis also investigated. Life intimidating CHF is the most sterning

form of anthracycline-induced cardiomyopathy and represents the cumulative

doselimiting toxicity of the drug.

Cutaneous

Reversible complete alopecia occurs in most of the cases. Hyperpigmentation of

nailbeds and dermal creases and onycholysis have been described in a several cases.

Rash, itching, or photosensitivity may also occur.

Gastrointestinal

Acute nausea and vomiting occurs frequently and severly and can be improved by

antiemetic therapy. Mucositis (stomatitis and esophagitis) may occur within 5 to 10

days after starting of therapy and most of the patients get well from this adverse

reaction within the another 5 to 10 days or may be severe leading to ulceration and

origin of severe infections.

Hematologic

As with other cytotoxic agents, doxorubicin may cause myelosuppression.

Myelosuppression requires cautious monitoring. Total and differential WBC, red

blood cell (RBC) and platelet counts should be considered before and during each

cycle of therapy with the drug doxorubicin. Clinical effects of severe

myelosuppression include fever, infections, sepsis, septic shock, hemorrhage, tissue

hypoxia, or death.



Hypersensitivity

Fever, chills and urticaria have been reported and it was considered that Anaphylaxis

can occur. A case of obvious cross sensitivity to lincomycin has been reported.

Neurological

Peripheral neurotoxicity in the form of local-regional sensory and/or motor

disturbances have been reported in patients treated intra-arterially with doxorubicin

and/or with cisplatin. In animal studies it was inestigated that; seizures and coma in

rodents and dogs treated with intra-carotid doxorubicin occurs.

Ocular

Keratitis, Conjunctivitis and lacrimation occur usually.


Matrial and Methods

CHAPTER 4

MATRIAL AND METHODS

List of Chamicals Used

Table No. 4.1 List of Chemicals Used.

S.No Chemicals

01 ETHYL ALCOHOL(AR)

02 Formaldhyde solution 10%

03 Rat feed

04 Sodiun chloride(ar)

05 Ketamine.HCL. inj. For i.m.,slow i.v.,i.v. infusion: Aneket

06 Anaethetic ether i.p.500 ml

LIST OF DRUGS USED

Table No. 4.2: List of Drugs Used.

S.No Drugs

01 Doxorubicin HCL

02 Boerhaavia Diffusa

03 Asparagus Racemosus

DOSE SELECTION AND PREPATION

Prepation of doxorubicin and dose selection

Doxorubicin Hydrochloride (DOX): Doxorubicin was supplied as a reddish orange,

hygroscopic in nature, crystalline powder form. DOX solution shoul be freshly

prepared and protected from light and was dissolved in saline solution. dosage of the

doxorubicin administration intraperitional was prepared in (0.9%) sodium chloride

saline solution.

Dose of doxorubicin each dose contining 2.5mg/kg body wt. for a cumalitive dose of

15 mg/kg body wt. for the present study, were chosen based on the previous reports.

Prepation of combination of harbal drug

Boerhaavia Diffusa(P) and Asparagus Racemosus(S) powder was suspended in 0.9%

saline sodium chloride and each animals belonging to two groups recieved 4ml of PS.

at a dose of 250 mg/kg body weight everyday respectively by the intragastric

intubation.

Route of administration: Oral and intrapenitional.


Matrial and Methods

EXPERIMENTAL ANIMALS

Wister albino rats of either sex weighing 150-200 grams, were procured from animal

house, Arya College of pharmacy, Jaipur. The animals were housed under standard

laboratory conditions of 25 ±2C temperature, relative humidity of 55% and 12:12 hour

light : dark cycle maintain during the study. The animal were given standred rat pellet

and tap ad libitum. The experimental protocol was approve by institutional animal

ethical committee(IAEC), committee for purpous of control and supervision of

experiments on animals (CPCSEA).

II. METHOD

Experimental Design And Protocol

Dox induced myocardial toxicity

Group I:- CONTROL (Control group): Animals were treated with normal saline

5ml/kg.

Group II:- DOX (Doxorubicin treated): Doxorubicin was administered

intraperitonially in each dose containing 2.5mg / kg of the body weight for a

cumulative dose of 15mg/kg of the body weight.

Group III: PS+DOX

Pretreated daily (200mg/kg/oral) for 2 weeks and after pretreatment then alternatively

with Doxorubicin injection (for a cumulative dose of 15mg/kg body weight) for next

2 weeks. At the last treatment, blood samples were obtained under ether anesthesia

using heparin treated micro capillaries for the estimation of enzyme biomarkers (ALP,

LDH and CPK). Control and treated animals was observed for a period of 4 weeks

after the last injection, for the appearance, behavior and mortality. At the end of 4

weeks post treatment period, animals were anaesthetized with anesthetic ether and

ECG parameters were recorded using BPL 05 cardiart. The animal were killed under

ether anesthesia and a midline abdominal incision was performed and heart tissue was

quickly dissect out, washed in cold normal saline, dried on filter paper and weighed.

Group IV: B.diffusa(P): was pretreated daily (200mg/kg/ oral) mix with 0.9% normal

saline for 2 weeks and then with doxorubicin alternatively for next 2 weeks.


Matrial and Methods

Group V: A.racemosus(S): S was pretreated daily (200mg/kg/ oral) with 0.9%

normal saline for 2 weeks and then with doxorubicin alternatively with normal saline

0.9% for next 2 weeks.

At the end of experiment, the animals were subjected to:-

Mortality: Death rate was determined.

Behaviour: Changes in behaviour was observed.

BODY WEIGHT PARAMETERS

Body weight of each rat was recorded every 15 days till the completion of 30 day

study.

Serum parameters

All the animals were fasted overnight but allowed to free access to water. on the last

day of drug administion, animals was anaesthetized with ether and blood sample was

collected by the retro-orbital plexus of each rat. then serum was seprated; Alkaline

phosphate (ALP), lactate dehydrogenase(LDH) and creatine phosphokinaase-MB

isoenzyam, (CPK-MB) were measured by kits or tested in pathology lab.

A. Estimation of Alkaline phosphate (ALP)

Clinical significance: The high concentrations of ALP in liver, bone, placenta,

intestine and certain tumours’.

Increases: High level of ALP, the enzyme occurs in liver diseases, bone diseases,

Hodgkin’s disease or congestive heart failure.

Decreases: Low levels of ALP occur in hypophosphatasia and malnourished patients.

B. estimation of lactate dehydrogenise (LDH)

Clinical significant

The enzyme LDH is concentrated in cardiac, renal, liver, muscle and other body

tissue.

When LDH increased in body then following disordered generate myocardial

infarction, kidney damage and other muscle disease.


Matrial and Methods

C. Estimation of Creatine Kinase (CPK)

Clinical significance: Creatine kinase is found in skeletal muscle, cardiac muscle and

cerebral muscle tissues.

Increases: CPK levels increases in body then cause myocardial infarction, acute

cerebra-vascular disease and muscular injury. Following a myocardial infarction, CK

activity begins to rise within 3 to 5 hours, peaks between 20 and 30 hours and returns

to normal by the third day.

ELECTROCARDIOGRAPHIC PARAMETERS

Fig. No. 4.1: Electrocardiogram.

ECG was recorded at the end of the treatment after the last dosing. BPL 05 cardioart

ECG machine was used to record and monitor ECG tracings. take one Rats from each

group were anesthetized with ketamine (aneket) anesthesia, needle electrodes were

inserted under the skin for the limb lead. For each ECG tracing ST, QT interval, QRS

complex and heart rate were measured.


Matrial and Methods

At the time of ecg recording ecg graph paper speed is 25 milliM/sec.

Heart rate: Heart rate canbe easily determined y the counting the number of large

squars between two consecutive QRS complexs. Ecg tracing covers 5 large squars or

one large squar is equal to 0.2 sec. one small squar is equal to 0.04 beat/sec. normal

heart rate is 60 to 100/min.

STATISTICAL ANALYSIS

All result are expressed as MEAN±S.E.M. statistical significance is calculated using

studdent’s t-test which was employed for comparison of control group and other test

groups.

P<0.05 was considered as significant.

P value less then 0.05 and greter then 0.01 is significant.

P value less then 0.01 and greater then 0.001 is very significant.

P value less then 0.001 is concidered to be extremely significant.


General Obesrvation and Mortality

CHAPTER 5

GENERAL OBESRVATION AND MORTALITY

A. GENERAL APPERANCE

In the general appearance all group of animals was throughout the study/experiment

period. In the experimental period animals are shows following characters by each

group.

1. Doxorubicin treated animals fur become scruffy, soft watery faces and enlargement

abdomen. This condition is more severe at the end of the experiment period. And the

other hand these types of symptoms are not observed in other group (B. Diffusa, A.

Racemosus, normal saline group and combination group.

2. Mortality

In the doxorubicin group 50% mortality observed. But there were no mortality

observed in the normal saline, B. Diffusa, A. Racemosus and there combination of

the.

Table No 5.1: Mortality Rate.

S.NO TREATMENT MORTALITY % VALUE

1 GROUP I (N.S) 0/6 0%

2 GROUP II(DOX) 3/6 50%

3 GROUP III (P+S+DOX) 0/6 0%

4 GROUP IV(P+DOX) 0/6 0%

5 GROUP V (S+DOX) 0/6 0%

(N.S-NORMAL SALINE, DOX-DOXORUBICIN, P-PUNARNAVA, S-

SATAVARI).

3. Doxorubicin treated animals showed gradually decreased in food and water

consumption, as compare to normal saline. But this consumption was improved in the

pre-treatment duration in each group.

Heart weight, body weight and ratio of heart and body

The result shown from table no. and fig.no...shows that the heart weight in the NS,

DOX, PS+D, P+D, S+D (n=6) treated rat were 0.76±0.004, 0.94±0.006,a,

0.77±0.003,ns

0.82±0.04 and 0.80±0.03gm, respectively. The heart weight in

doxorubicin treated rat was significantly increased compare with normal rat. The heart



weight combination of b.diffusa and aspergus racemosus was not significant as

compared with normal rat. The heart weight in pre treated group i.e. PS+D, P+D, S+D

was significant decreased in compare with doxorubicin treated group.

The body weight in NS, DOX, PS+D, P+D, S+D (n=6) treated rats were 250.66±4.13,

157.66±5.18, 231.16±3.76, 202.66±4.76 and 201±4.46.

The body weight of doxorubicin treated rats is significantly decreased compare with

normal rats. The body weight of pre treated group rats is increased. And compare with

doxorubicin weight is significantly increased.

The ratio of heart weight and body weight in doxorubicin treated rat is significantly

increased as compare with normal rats. The ratio of PSD, PD and SD is not significant

compare with normal rats. And these group are compare with doxorubicin group they

are significantly decreased.

Table No. 5.2: Heart Weight, Body Weight, Andheart / Body Weight Ratio.

S.NO TREATMENT BODY

WEIGHT(gm)

HEART

WEIGHT(gm)

RATIO OF H/B

WEIGHT(x10-3

)

01 NS 250.66±4.13 0.76±0.004 3.031

02 DOX 157.66±5.18a 0.94±0.006

a 5.962

a

03 PSD 231.16±3.76ns

0.77±0.003ns

3.331ns

04 PD 202.66±4.76 0.82±0.04 4.046

05 SD 201.5±4.46 0.80±0.03 3.970

Value are mean ± SEM; n=6 in each group, a p<0.01 when compare to normal.

Ns= not significant, p<0.01 when compare to doxorubicin.

P value less then 0.05 and greter then 0.01 is significant.

P value less then 0.01 and greater then 0.001 is very significant.

P value less then 0.001 is concidered to be extremely significant.



Fig. no. 5.1: Body weight of animal model.

Figure 5.2: Heart weight of animal model.

Estimation of enzyme biomarker

ALP, CK, LDH

It’s depicted from table no. and fig. no. that the ALP level in the NS, DOX, PS+D,

P+D, S+D. (n=6). Treated rats were 142.16±3.43, 270.83±5.74, 216.83±5.19,

199.83±5.38 and 207.33±5.08 respectively.

It is evident from table no... And the fig.no...that the CK level of NS, DOX, PS+D,

P+D, S+D treated rat were 2607±6.44, 3011.16±7.41, 2691.66±6.21, 2729.83±3.43,

2753.5±5.12 respectively.



It is evident from table no. And the fig.no. Observation for the LDH level of NS,

DOX, PS+D, P+D, S+D treated rats were 152.33 ± 6.68, 357.5 ± 6.44, 307 ± 6.87,

207.66 ± 5.50 and 285.5 ± 5.4 respectively.

Doxorubicin treated rats shown a significant increased in the levels of ALP, CK, LDH

as compare to normal group.

Table no. 5.3: Estimation of ALP, CK and LDH (Normal).

S.NO ALP(IU/L) CK(IU/L) LDH(IU/L)

01 142 2610 153

02 147 2602 146

03 138 2615 163

04 139 2598 147

05 145 2612 157

06 142 2605 148

MEAN±SEM 142±3.43 2607±6.44 152.33±6.68

Table no. 5.4: Estimation of ALP, CK and LDH (DOX).


01 273 3015 350

02 279 2998 356

03 269 3018 360

04 275 3009 359

05 268 3017 368

06 265 3010 352

MEAN±SEM 270.83±5.74 3011.16±7.41 357.5±6.44

Table no. 5.5: Estimation of ALP, CK and LDH (PS+D).


01 216 2695 310

02 210 2690 316

03 218 2688 298

04 213 2696 305

05 219 2682 301

06 225 2699 312

MEAN±SEM 216.83±5.19 2691.6±6.21 307±6.8

Table no. 5.6: Estimation of ALP, CK and LDH (P+D).


01 198 2730 210

02 204 2725 215

03 194 2732 200

04 208 2728 207

05 195 2735 203

06 200 2729 211

MEAN±SEM 199.83±5.38 2729.83±3.43 207.66±5.5



Table no. 5.7: Estimation of ALP, CK and LDH (S+D).


01 210 2756 280

02 205 2750 289

03 214 2758 278

04 201 2748 285

05 211 2760 291

06 203 2749 290

MEAN±SEM 207.3±5.08 2753.5±5.1 285.5±5.4

Table no. 5.8: Comparetive Study of Serum Enzymes Biomarkers.

S.NO TREATMENT ALP(IU/L) CK(IU/L) LDH(IU/L)

01 NS 142±3.43 2607±6.44 152.33±6.68

02 DOX 270.83±5.74 3011.16±7.41 357.5±6.44

03 PS+D 216.83±5.19 2691.6±6.21 307±6.8

04 P+D 199.83±5.38 2729.83±3.43 207.66±5.5

05 S+D 207.3±5.08 2753.5±5.1 285.5±5.4

P.VALUE P<0.0001 P<0.0001 P<0.0001

Figure 5.3: Graph of ALP of treated rats in myocardial toxicity.



Figure 5.4: Graph of CK of treated rats in myocardial toxicity.

Figure 5.5: Graph of LDH of treated rats in myocardial toxicity.

ELECTROCARDIOGRAPHIC ANALYSIS

Electrocardiograph takes at the end of experiment, in ECG various types of change

shown. In different treatment groups, Due to the effect of drug treatment some

changes in the ST segment, QT interval and QRS complex.



Figure 5.6: Normal Saline (Ns) Treated Rat.

In the normal saline treated rat groups ECG consist of P, QRS and T waves. the onset

of arterial activation to the ventricular activation is calculated by PR interval. The

conduction pathways through the ventricules are represented by the QRS complex.

And at the time interval during the ventricular myocardium is depolarized and

isoelectric line is normal conditions is denoted by ST segment.

Figure 5.7: Doxorubicin (Dox) Treated Rat.



Intraperitoneal injection of doxorubicin may affect decreased in the heart rate and

show the alteration in the PQRST waves. A lead II ECG show change in ECG and

includes shortened QT interval, prolong PR interval, and prolong QRS interval.

Increased QRS voltage, flattening T waves. And notching QRS waves, that shows

atrial and ventricular fibrillation. Due to doxorubicin desirable arrhythmias, this

shows acute toxicity of dox.

Figure 5.8: Combination OD B.Diffusa and Aspergus Racemosus and

Doxorubicin (Ps+D) Treated Rat.

Figure 5.9: Boarheebia Dffusa and Doxorubicin (P+D) Treated Rat.



Figure 5.10: Aspergus Racemosus and Doxorubicin (S+D) Treated Rat.

Upon administration of herbal drug (orally), administration of combination of herbal

drugs (orally) and administration of doxorubicin in animal body. The herbal drug

normalizes the PQRST waves and hence atrial and ventricular fibrillation. Also the

heart rate was increased as compare to doxorubicin treated group.

Table 5.9: Heart Rate Analysis.

S.NO ANIMAL GROUP HEART RATE(beats/min)

01. NS 323.33±6.31

02 DOX 168.83±4.26

03 PS+D 252.84±4.96

04 P+D 261.16±7.80

05 S+D 258.15±5.91



Figure 5.11: Comparision of heart rate of different group of animal.


Discussion

CHAPTER 6

DISCUSSION

Doxorubicin-induced cardiac toxicity changes the group of the cardiomyocyte and

induces apoptosis, which is a potentially modifiable and preventable form of

myocardial tissue loss.[217]

This potentially novel mechanism is transient, but it may

be of a key importance to the ensuing heart failure. The results of this study have

confirmed that a single dose of dox (15 mg/kg) induces acute cardiac toxicity in rats.

This is in conformity with previous studies 218. Doxorubicin induced myocardial

toxicity have been well documented in experimental animal. The present study was

done to investigate the influence of Boerhavia diffusa and Asparagus Racemosus and

their combination on Dox-induced cardiotoxicity in albino rats. Following lines of

evidence can be emphasized from the present study.

1) General observation

A) In Dox treated group following changes were observed

The animal fur becomes scruffy.

Necrosis observed at the site of Dox injection.

There was 50% mortality in Dox treated group.

Animal becomes lazy.

B) In pretreated group with B. Diffusa, A. Racemosus and their combination.

No necrosis observed.

No any mortality.

No effect on animal activity.

2) Heart and body weight

The Dox treated rats showed increase in the heart weight and also decrease in body

weight. The body weight may be decreased due to the reduced intake of food. And the

heart weight may be increased due to the loss of myofibrils, increased number of

Liposomes, mitochondrial swelling, sarcoplasmic reticulum dilation. The gross

anatomically change of the heart treated with Doxorubicin showed a typical chronic

toxic response including cardiac hypertrophy and overall enlargement of heart. That

was determined by heart weight and ratio of heart weight and body weight.


Discussion

3) Serum enzyme biomarker

The study reveals rigorous biochemical changes with oxidative injury in the cardiac

tissue after the chronic treatment with Doxorubicin (cumulative dose of 15mg/kg).

Doxorubicin is a well known cardiac toxic drug due to its ability. It will damaged

myocardial cells. As a result of this, ALP, Lactate Dehydrogenase (LDH) and

Creatine kinase (CK) was released into the blood stream and provided as the

diagnostic markers of myocardial tissue damage.[219,220,221]

The quantity of these

cellular enzymes present in the blood reflects the alteration in plasma membrane

integrity and permeability.

In present study the biochemical changes as well as oxidative damage in cardiac

tissue after the treatment of cumulative dose of 15mg/kg of body weight with

Doxorubicin. Doxorubicin is a cardiac toxic agent because of its destructive ability for

myocardial cells because of which ALP, Lactate Dehydrogenase (LDH) and

Creatinine Kinase (CK) were released into blood stream and serve as the diagnostic

biomarker of myocardial tissue damage. The amount of these cellular enzymes in

blood effects the alteration in plasma membrane integrity and permeability.

In this study, Doxorubicin treated shows the significant elevation in the level of these

diagnostic biomarker enzymes (ALP, CK and LDH). Moreover, elevated levels of

these enzymes are indicator of the severity of Doxorubicin induced cardiotoxicity.

The prior administration of combination of B. Diffusa and A. Racemosus showed

significant reduction in Doxorubicin induced elevated serum biomarker enzymes.

This reduction in the enzyme level confirms that combination of the same is

responsible for maintenance of normal structural cardiac myocytes.

4) Electrocardiographic parameters

Electrocardiograph abnormalities are the main criteria generally used for the definite

diagnosis of the myocardial injury. The study shows a significant variation of ECG

patterns on Dox administered rats as compared with the normal control rats. The

attributed findings were elevation of the ST segment, reduction in the P waves, in

QRS complex and R-R interval. In addition there was a prolongation of the QT

interval. Furthermore ECG changes are the indicators of the severity of Doxorubicin

induced the myocardial damage and which is in line with an earlier report. The

successive loss of cellular membrane damage due to oxidative stress might be


Discussion

characterized by ST elevation. Treatment of combination of B. diffusa showed a

protective effect against Doxorubicin induced changed ECG parameter and eliminated

the acute fatal complication by protecting the cell membrane damage.

The data of the present study clearly show that combination of B. Diffusa and A.

Racemosus, modulated most of the electrophysiological, biochemical and

histopathological parameters were maintained to near normal status in Dox treated

rats, suggesting the beneficial action of combination of B. Diffusa and A. Racemosus

as a cardiac protective agent.


Summery and Conclusion

CHAPTER 7

SUMMERY AND CONCLUSION

In the present study we observed the effect of combination of B.Diffussa and A.

Racemosus on doxorubicin induced cardio-toxicity in rats. The result suggest that in

doxorubicin treated animals, significant decrease in the body weight and increased in

the heart weight and heart and body weight takes place. There was 50% mortality

found in the doxorubicin group. but in the pre-treated group no mortality found. And

body weight increased and heart weight decreased, the observation in the results

suggests that the combination of B.diffusa and A.racemosus significant the dox

induced free radicals mediated cardio-toxicity.

The data collected in the present study show that the cumulative administration of

doxorubicin increased the serum cardio-toxicity indices ALP, CK, LDH activity.

According to experiment result the combination of B.Diffusa and A.Racemosus

protect the myocardium from doxorubicin mediated damage as evident from the near

normal value of these markers enzymes. This can be show as higher oxidative stress

in the cardiac tissue from rats following a cumulative dose of doxorubicin and this

enhance oxidant injury was prevented by combination therapy.

Electrocardiograph abnormalities are the main criteria generally used for the define

diagnosis of myocardial injury. The experiment show signification alterations of ECG

pattern were observed in the doxorubicin administrated rats as compare to normal

control rats. The characteristic finding were elevation of ST segment, reduction of P

segment, QRS complex and R-R interval. In addition there was prolongation of QT

interval. The consecutive loss of cellular membrane damage due to oxidative stress

might be characterized by the ST elevation, combination of b.diffusa and a.recimosus

administration shown a protective effect agenised the doxorubicin induced alter ECG

parameters and eliminated the acute fatal complication by protecting the cell

membrane damage.

The enhance generation of oxygen radicals may directly damage cellular structure as

seen in histological slides. Pre treatment with combination of B.Diffusa and

A.Racemousu effectively inhibits doxorubicin induced cardiac damage by reversal of

infiltration of inflammatory cell and fragmentation of myofibrils.


Summery and Conclusion

Collectively these all parameters as electrocardiographic, biochemical, result provide

a possible and potential cardioprotection against doxorubicin cardio-toxicity.

Finally we concluded that the cardio-toxicity induced by the doxorubicin is in

relationship with oxidative stress and biochemical changes in heart cells and tissues.

Combination of B.Diffusa and A. Racemosus has shown to be most effective in

functional recovery of the heart and restoring the biochemical and histopathological

alteration which may be associated with its potent antioxidant property. This

experiment suggested that combination of B. Diffusa and A. Racemousu may be

considered as a potentially useful drug in the combination with doxorubicin to limit

free radical mediated the organ injury. Additionally molecular level of the

investigation is to be done using different animal’s modal and using different

biochemical parameters to assess the possible mode of action of B.Diffusa and A.

Racemosus as cardio-protective agent.


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01 introduction 1-4 02 aims and objective 5 03 reviwe of

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