Dissertation Dhiraj 2012

8/13/2019 Dissertation Dhiraj 2012

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Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in

Charles Foster rats . Page 1

LIST OF ABRIVIATIONS:

l---------- Micro liter

CPA -------Cyclophosphamide

NC--------- Normal control

SGPT..Serum glutamic pyruvate transaminase

SGOT.Serum glutamic oxaloacetic transaminase

LS Longitudinal section

TS . Transverse section


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INTRODUCTION:

Cyclophosphamide (CP), is a widely used cytotoxic alkylating agent with antitumor and

immunosuppressant properties. It is used for the treatment of chronic and acute leukemia,

multiple myeloma, lymphomas, rheumatic arthritis and systemic lupus erythematosus and

in the preparation for bone marrow transplantation(Dollery , 1999). Cyclophosphamide

undergoes bioactivation by the hepatic microsomal cytochrome P450 mixed function

oxidase system to active metabolites that enter the circulatory system. Phosphoramide

mustard and acrolein are the two active metabolites of cyclophosphamide (Ludeman,

1999). The antineoplastic effects of cyclophosphamideare associated with phosphoramide

mustard, whereas acrolein is linked to toxic side-effects like cell death, apoptosis, oncosis

and necrosis (Kern etal.,2002). In spite of its therapeutic importance, a wide range of

adverse effects including reproductive toxicity has been demonstrated following

cyclophosphamide treatment in humans and experimental animals (Anderson, et al.

1995). Adult male patients treated with CP have demonstrated diminished sperm counts

and an absence of permatogenic cycles in their testicular tissue (Howell, Shalet S. 1998).

Previous studies on male rats have confirmed the potential of CP to cause oligospermia,

azoospermia and histological alterations in the testis and epididymis (Meistrich , Parchuri

, Wilson , et al. 1995 , Kaur , et al. 1997) Decrease in weight of reproductive organ,

impaired fertility, growth and development of next generation was also observed in

cyclophosphamide treated male rats (Trasler et al ., 1986). Although the precise

mechanism by which CP causes testicular toxicity is poorly understood, numerous studies

have shown that CP exposure can disrupt the redox balance of tissues leading to oxidative

stress (Das etal., 2002). It has been reported that oxidative DNA damage is caused by

hydroperoxide derivative of CP through generation of H2O2 (Murata et al., 2004).

Further, spermatozoa are more susceptible to peroxidative damage because of high

concentration of polyunsaturated fatty acids and low antioxidant capacity (Vernet, et al.,

2004). Also, acrolein has been found to interfere with the tissue antioxidant defense

system and produces highly reactive oxygen free-radicals that are mutagenic to

mammalian cells (Arumugam , et al. ,1997 ) Consequently, from these aforementioned


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studies, combination of the drug delivery together with potent and safe antioxidant may

be the appropriate approach to reduce CP-induced reproductive toxicity.

Properties

Cyclophosphamide is an antineoplastic and immunosuppressant agent that is usually a

fine white crystalline powder at room temperature. The substance liquefies and becomes

an oily semisolid mass when water is removed under high vacuum. It is soluble in water,

alcohol, chloroform, dioxane, and glycols, slightly soluble in benzene and carbon

tetrachloride, very slightly soluble in ether and acetone, and insoluble in carbon disulfide.

Cyclophosphamide is sensitive to oxidation, moisture, and light (Akron 2009).Physical

and chemical properties of cyclophosphamide are listed in the following table.

(RS)-N,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide

Property Information

Molecular weight 261.1a

Density 1.479 g/cm3b

Melting point 49.5C to 53Ca

Boiling point 336C b

LogKow 0.63a

Water solubility 40 g/L at 20Ca

Vapor pressure 4.45 105 mm Hg at 25oc
http://en.wikipedia.org/wiki/File:R-cyclophosphamide-from-xtal-1996-3D-balls.pnghttp://en.wikipedia.org/wiki/File:Cyclophosphamid.svghttp://en.wikipedia.org/wiki/File:R-cyclophosphamide-from-xtal-1996-3D-balls.pnghttp://en.wikipedia.org/wiki/File:Cyclophosphamid.svg


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Indications

Cyclophosphamide is used in the treatment of chronic lymphocytic leukaemia,

lymphomas, soft tissue and osteogenic sarcoma, and solid tumours. It is given orally or

intravenously. Cyclophosphamide is inactive until metabolized by the liver.

(a)Hodgkin lymphomaCyclophosphamide is used in combination regimens (e.g. bleomycin, etoposide,

doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone [known as

BEACOPP]) for the treatment of Hodgkin lymphoma.

(b)Non-Hodgkin lymphomaCyclophosphamide is used in combinationtherapy for the treatment of non-Hodgkin

lymphoma, including high-grade lymphomas, such as Burkitt lymphoma and

lymphoblastic lymphoma, as well as intermediate- and lowgrade lymphomas.

Cyclophosphamide is commonly used with doxorubicin (hydroxydaunorubicin),

vincristine (oncovin), and prednisone (known as the CHOP regimen), with or without

other agents, in the treatment of various types of intermediate-grade non-Hodgkin

lymphoma.Cyclophosphamide has also been used as a single agent in the treatment of

low-grade lymphomas.

(c)Multiple myelomaCyclophosphamide is used in combination with prednisone, or as a component of

combination chemotherapy (i.e. vincristine, carmustine, melphalan, cyclophosphamide,

and prednisone [VBMCP]) for the treatment of multiple myeloma.

(d)LeukaemiaCyclophosphamide is used commonly for the treatment of chronic lymphocytic

(lymphoblastic) leukaemia. Cyclophosphamide is used in combination with busulfan as a

conditioning regimen before allogeneic haematopoietic progenitor cell transplantation in

patients with chronic myelogenous leukaemia.Cyclophosphamide is used in the treatment

of acute lymphoblastic leukaemia, especially in children. In the treatment of acute

myeloid (myelogenous, non-lymphocytic) leukaemia, cyclophosphamide has been used

as an additional drug for induction or post-induction therapy.


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(e)Cutaneous T-cell lymphomaCyclophosphamide is used alone or in combination regimens for the treatment of

advancedmycosis fungoides, a form of cutaneous T-celllymphoma.

(f) NeuroblastomaCyclophosphamide alone is used in the treatment of disseminated neuroblastoma.

Combination chemotherapy that includes cyclophosphamide is also used for this

neoplasm.

(g)Cancer of the ovaryCyclophosphamide is used in combination chemotherapy (vincristine, actinomycin D,

and cyclophosphamide [VAC]) as an alternative regimen for the treatment of ovarian

germ-cell tumours. Cyclophosphamide has been used in combination with a platinum-

containing agent for the treatment of advanced (Stage III or IV) epithelial ovarian cancer.

(h)RetinoblastomaCyclophosphamide is used in combination therapy for the treatment of retinoblastoma

(i) Cancer of the breastCyclophosphamide is used alone and also in combination therapy for the treatment of

breast cancer. Combination chemotherapy with cyclophosphamide is used as an adjunct

to surgery in premenopausal and postmenopausal women.

BRIEF RESUME OF THE INTENDED WORK:

NEED FOR STUDY:

Cancer is the leading cause of death in economically developed countries and the

second leading cause of death in developed countries (Isselbacher et al,1994.) According

to 2011 cancer prevalence in India it is estimated to be around 2.5 million with over

80,0000 new cases and 5,50,000 death occurring each year due to this disease

Chemotherapy is the primary treatment available for disseminated malignant disease,

most common chemotherapy agents which acts by killing the cells that divides rapidly,

one of the main properties of cancer cell. This means chemotherapy also harms the cells

that divide rapidly under normal circumstances in bone marrow (Isselbacher et al,1994.)


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Immunosuppression is the major drawback in chemotherapy and it also has the toxic side

effects like myelosuppression, mucosal ulceration and alopecia etc (Leemol et al.)

Cyclophosphamide is one of the most widely used broad spectrum antitumor agent it is

used in the treatment of carcinoma of breast, lungs, ovary, bladder, non Hodgkins, acute

and chronic leukemia etc.

Cyclophosphamide itself carcinogenic, potentially carry transitional cell carcinoma of

bladder as a long term complication, it can lower the bodys ability to fight an infection

causing Immunosuppression and also have side effects like bone marrow toxicity and

testicular cell damage.

Cytoprotection means protection of cell from noxious chemicals or other stimuli

or Enhancing the ability of cell to resist injury. (Dorlands.et al, 2000)

Cytoprotective agents will reduce or prevent these toxicities, the agents should

ideally be selective for normal cell versus cancer cells, these are effective in reducing or

preventing toxicity should have no negative impact on anticancer therapy and have

minimal adverse effect.

ASHWAGANDHA AS AN IMMUNOMODULATER

Withania somnifera Dunal (ashwagandha, WS) is widely used in Ayurvedic medicine,

the traditional medical system of India. It is an ingredient in many formulations

prescribed for a variety of musculoskeletal conditions (e.g., arthritis, rheumatism), and as

a general tonic to increase energy, improve overall health and longevity, and prevent

disease in athletes, the elderly, and during pregnancy. (Duke, 1985) Many

pharmacological studies have been conducted to investigate the properties of

ashwagandha in an attempt to authenticate its use as a multi-purpose medicinal agent. For

example, anti-inflammatory properties have been investigated to validateits use ininflammatory arthritis, (Bone K. 1996, Wagner H, et al. , 1994 , Anabalgan et al. 1981)

and animal stress studies have been performed to investigate its use as an antistress agent.

(Bhattacharya et al, 1995, Bone .1996) Several studies have examined the antitumor and

radio sensitizing effect of WS (Devi, et al, 1993). The purpose of this paper is to review


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the literature regarding WS and report on clinically relevant studies, in an attempt to

establish a scientific basis for the therapeutic use of WS. Results of studies investigating

the chemistry and toxicity of WS will also be discussed.

Studies indicate ashwagandha may have benefits of anti-inflammatory,antitumor,

antistress,antioxidant, immunomodulatory, hemopoietic, and rejuvenating effects. It also

appears to exert a positive effect on the endocrine, cardiopulmonary, and central nervous

systems. Some researchers suggest Ashwagandha exhibits a variety of benefits with

Limited side effects or toxicity (Mishra et al.,2000). Ashwagandha, used in traditional

Indian and Ayurvedic medicine, grows in India and Africa. The roots of

Ashwagandha are believed to have health benefits on various conditions including

inflammation (including arthritis), and a wide range of infectious diseases. (Duke, 1985)

Ashwagandha contains withanolides as its major active ingredients to account for most of

its

medicinal benefits or uses. (Wagner, 1994) Basic studies have shown its ability to

simulate the immune system cells, inhibit inflammation and improve memory in animal

studies. Thus, it is not a surprise that herbalists claim ashwagandha as a tonic or

adaptogen. It may relieve anxiety. Adaptogen is an herb counteracts the effects of stress

and promotes general wellness. Usually, marketers suggest dosages of 3-6 grams of the

dried root a day.


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REVIEW OF LITERATURE

BACKGROUND

Cyclophosphamide is an antineoplastic compound that is chemically related to nitrogen

mustard. Cyclophosphamide is an odorless, fine white to off-white crystalline powder

that is soluble in water and ethanol (NTP, 2005). Cyclophosphamide is used clinically to

treat a wide range of cancers including malignant lymphomas, myeloma, leukemia,

mycosis fungoides, neuroblastoma, adenocarcinoma, retinoblastoma, and breast

carcinoma (Bristol-Myers Squibb Co, 2003). Other clinical uses for CPH include

immunosuppressive therapy following organ transplants or as a treatment for

autoimmune disorders such as rheumatoid arthritis, Wegeners granulomatosis, andnephritic syndrome in children (Chabner et al., 2001). Metabolism of CPH takes place in

the liver and undergoes metabolic activation by cytochrome P450 isoenzyme 2B

(Chabner et al., 2006). The major circulating metabolite of CPH, 4-

Hydroxycyclophosphamide, is in equilibrium with its tautomer, aldophosphamide, which

is spontaneously broken down to produce phosphoramide mustard and acrolein (Zhang et

al., 2005). Phosphoramide mustard is responsible for anti-tumor effects, while acrolein is

responsible for the hemorrhagic cystitis observed during CPH therapy (Chabner et al.,

2006). Cyclophosphamide is a known alkylating agent with alkylating properties that

result in nucleotide base mispairs and DNA/DNA or DNA/protein cross-linking that lead

to major disruptions in nucleic acid function and the inhibition of DNA synthesis (Zhang

et al., 2005). Cyclophosphamide-induced nucleic acid damage may lead to DNA

mutations that result in cytotoxicity, carcinogenicity, teratogenecity, and reproductive

toxicity following chronic exposure to CPH (NTP, 2005; Gilian and Charzinoff, 1983;

Mirkes, 1985; Meirow et al., 2001). The negative health effects associated with CPH

present a significant health and safety threat to laboratory staff, animal handlers, and

other personnel who may be subject to accidental exposure. Due to this health and safety

threat the Institutional Biosafety Committee (IBC) has classified CPH as a reportable

hazardous chemical that must be registered on the Institutional Animal Care and Use

Committee (IACUC) protocol Appendix C for Chemical Hazards.


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Occupational Exposure Hazards:-

Primary routes of occupational exposure to CPH include: inhalation, accidental injection,

and dermal absorption (NIOSH, 2004; NTP, 2005). A limited number of studies have

examined chronic health effects related to occupational exposure to CPH and have

reported an increased incidence of cancer among health care workers (Sessink et al.,

1993). However, chronic effects in patients treated with CPH are well documented. The

available scientific literature indicates that chronic long-term exposure to CPH could lead

to a number of serious health effects.

1. Carcinogenicity: Cyclophosphamide is a known alkylating agent that has been

sufficiently studied in a variety of in vivo and in vitro assays (NTP, 2005). In host-

mediated assays, CPH induces chromosomal aberrations, sister chromatid exchange, and

gene conversions (IARC, 1987). Laboratory animals exposed to CPH by various routes of

administration develop benign and malignant tumors of the bladder, breast, lungs, liver,

and injection site (IARC, 1981). In addition, rats treated with CPH developed leukemia

and lymphoma (IARC, 1981 and 1987). Several epidemiological studies have

consistently found excesses of bladder cancer and leukemia among people treated with

CPH for various medical conditions (IARC, 1981; Kinlen, 1985; Pedersen-Bjergaard et

al., 1985; Greene et al., 1986; Haas et al., 1987). Cyclophosphamide is classified Group 1by IARC (1981), as a known human carcinogen.

2. Cytotoxicity: The cytotoxic effects of CPH are generally considered to be the result of

DNA crosslink formation through covalent bonding of highly reactive alkyl groups of the

alkylating nitrogen mustards (Zhang et al 2005). The alkylation of the 7-nitrogen atom of

guanine in DNA molecules takes place by phosphoramide mustard resulting from CPH

activation (Pette et al., 1995). At alkaline or neutral pH, the nitrogen mustard is converted

to chemically reactive carbonium ion through imonium ion. Carboinium ions react with

the N7 of guanine residues in DNA to form a covalent linkage. The second arm in the

phosphoramide mustard can react with a second guanine moiety in an opposite DNA

stand or in the same stand to form crosslinks (Fleer and Brendal, 1983; Springer et al.,

1998). Following crosslink formation, the cells will undergo apoptosis initiated by DNA

damage and inhibition of DNA replication, modulation of cell cycle, and other anti-


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proliferative effects (Bhatia et al., 1995; Chien and Ashman, 1986; Crook et al., 1986;

Masta et al., 1995; OConnor et al., 1991).

3. Teratogenicity: Cyclophosphamide is clearly teratogenic in animals with similar

mutations reported in multiple laboratory animal species (Gilani and Charzinoff, 1983;

Mirkes, 1985). Cyclophosphamide teratogenicity is characterized by central nervous

system, skeletal, and facial anomalies (Gilani and Charzinoff, 1983; Mirkes, 1985;

Padmanabhan and Singh, 1984). In addition, CPH is a known human teratogen with a

recognizable pattern of malformation known as Cyclophosphamide Embryopathy (Vaux

et al., 2003). Human malformations include growth deficiencies (pre- and postnatal) and

central nervous system, facial, and skeletal anomalies (Vaux et al., 2003). Like the

carcinogenic effects of CPH, the teratogenic effects are mediated through alkylating

intermediates, phosphoramide mustard and acrolein (Mirkes, 1985; Stahlmann et al.,

1985).

4. Reproductive Toxicity: Cyclophosphamide is associated with reproductive toxicities

in both males and females (Bristol-Myers Squibb, 2003; Wetzels, 2004). Both

spermatogenesis and oogenesis are interrupted following treatment with CPH (Wetzels,

2004). Cyclophosphamide induced sterility is dependent upon dose, duration of exposure,

and the state of gonadal function at the time of exposure (Bristol-Myers Squibb, 2003;

Wetzels, 2004). In females, amenorrhea has been associated with CPH exposure due to

decreased estrogen and increased gonadotropin secretions (Wetzels, 2004). Late

prepubescent females have developed ovarian fibrosis with complete loss of germ cells

after prolonged CPH treatment (Bristol-Myers Squibb, 2003). Males exposed to CPH

may develop oligospermia or azoospermia associated with increased gonadotropin

release

(Bristol-Myers Squibb, 2003; Wetzels, 2004). Cyclophosphamide induced reproductive

toxicities may be reversible (Bristol-Myers Squibb, 2003).


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Absorption, distribution,metabolism, and excretion:-

In most species, cyclophosphamide is rapidly absorbed, metabolized, and excreted. Its

metabolic pathway has been studied in several speciesincluding mice, rats, hamsters,

rabbit, dogs, sheep, and monkeys. Cyclophosphamide is notcytotoxic per se, because it

requires metabolic activation before it can act as an alkylatingagent. Activationtakesplace

predominantly in the liver, although this may occurin other tissues (IARC,1981).

Cyclophosphamide undergoes metabolism to several intermediates with

alkylatingactivity. The principal metabolites identifiedare phosphoramide mustard, and

acrolein.Phosphoramide mustard can undergo dephosphoramidationto yield nornitrogen

mustard,which also hasalkylating activity. Metabolites of cyclophosphamide can interact

with DNA and proteins, resulting in the formation of adducts .The metabolism ofcyclophosphamide and DNA adducts formation are summarized in Fig. minor pathway

results in dechloroethylationand the formation of dechloroethylcyclophosphamideand

another alkylating agent,chloroacetaldehyde (Balu et al., 2002).The other compounds

such as 4-ketocyclophosphamide and propionic acid derivative arerelatively non-toxic,

and are the major urinarymetabolites of cyclophosphamide in severalspecies (IARC,

1981)

Genetic and related effects:-

I nteraction with DNA

Using 4-hydroperoxycyclophosphamide as an activated form of cyclophosphamide,

Mirkeset al. (1992) identified by mass spectrometric analysis the formation of the

monofunctionaladduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (nor-G) and the

bifunctional adductN,N-bis[2-(7-guaninyl)ethyl]amine (G-nor-G)in rat embryos in in-

vitro culture. The monofunctional adduct N-(2-hydroxyethyl)-N-[2-(7-

guaninyl)ethyl]amine (nor-G-OH) was detected in bladder tissue of rats injected with

[3H] cyclophosphamide (Benson et al., 1988). Using 32P-postlabelling analysis, a

phosphotriester was shown to be formed: (1) when phosphoramide mustard was reacted

with deoxyguanosine 5-monophosphate, (2) when cyclophosphamide was incubated with

calf thymus DNA in the presence of reconstituted cytochrome P450 (CYP) metabolizing


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system, and (3) in liver DNA from mice injected intraperitoneally with

cyclophosphamide (Maccubbin et al.,1991). Nornitrogen mustard reacts with guanosine

and with guanine bases in DNA to form nor-G initially, but this is converted to a

hydroxylated derivative (nor-G-OH), and to a crosslinked (between guanines) adducts

(G-nor-G) (Hemminki, 1987). Both monofunctional adducts, but not the cross-linked

adduct, were also detected when phosphoramide mustard was reacted with DNA (Cushnir

et al., 1990). Acrolein reacts with DNA to form O6-(n-propanalyl) guanine, and the

product of chloroacetaldehyde reaction with DNA is O6-(ethanalyl) guanine (Balu et al.,

2002). Acrolein can produce exocyclic adducts in DNA, including 1,N2-

hydroxypropanodeoxyguanosine and 1,N6-hydroxypropanodeoxyadenosine (Chung et

al., 1984; Foiles et al., 1990; Smith et al., 1990).The former was detected in

acroleintreatedhuman fibroblasts and in peripheralblood lymphocytes of a dog treated

with cyclophosphamide(Wilson et al., 1991). Nornitrogen mustard also reacts covalently

with proteins, and a method for the detection of cysteine-34 residue adducts in human

serumalbumin has been described (Noort etal., 2002).The single-cell gel comet assay is

used to detect single-strand breaks and other alkali-labilelesions in DNA exposed to

cyclophosphamide

1.Withani a sominif era(Ashwagandha) With Therapeutic Value

FIG:WITHANIA SOMINIFERA


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Ashwagandhaor Indian winter cherryis one of the most important herbs in ayurvedic

system of medicines and one of the most widely used Indian medicinal plant throughout the

world. Because of its vast area of application, ayurvedic physicians have used it extensively in

wide range of therapeutic indications. Due to its properties, it has gained a lot of respect in the

eyes of herbal healers. Ashwagandha, scientifically known as Withania somnifera Dunal is

often called as Indian ginseng. However, many studies have concluded that Ashwagandha is

therapeutically superior to Ginseng. Moreover, we do not find Ginseng-abuse kind of

syndrome with Ashwagandha. Ginseng may not be advisable to administer in pediatric age

group. Ginseng may be contraindicative in hypertension, insomnia, renal and cardiac

disorders, where as Ashwagandha has no such contraindications.

Traditionally from Vedic period (5000 years back) to present era, Ashwagandha is used as a

Rasayana (Rejuvevative & Reparative) for longevity, Balya (to give strength) for general

health promotion and as a Vajikarana (Aphrodisiac) to improve vigor and vitality. The

prolonged and continued usage of this herb since ages also exhibits its safety, tolerability and

efficacy profile. Various clinical trials and animal research support its use in stress, anxiety,

cognitive impairment, neurological disorders (Alzheimers and Parkinsons disease),

inflammation, emaciation, and erectile dysfunction and infertility conditions. Ashwagandhas

chemo preventive and immunomodulatory property makes it a potentially useful adjunct for

patients undergoing radiation and chemotherapy. Apart from its anti-oxidant property,

Ashwagandha boosts the immunity and is potential in debility due to stress.

Ashwagandha is a natural anabolic agent that helps in building lean muscle, increases

energy and stamina and reduce muscle breakdown during pronounced strenuous activity.

Extensive clinical and experimental studies indicate that ashwagandha possesses the following

properties: Anti stress, Immunomodulatory, Rejuvenative, Anti-oxidative, Anti inflammatory,

Anti-tumor, Erythropoetic. The major medicinal properties of this herb are confined to roots.

Ayurveda advocates the usage of roots alone for the above said properties. The leaves of this

herb are generally not administered orally and are used externally in inflammatory conditions.


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The name comes from the peculiar odor of this herb, a smell similar to that of a sweaty horse.

Taxonomic Classification:-

Kingdom Plantae(plants)

Sub kingdom Tracheaobionta (vascular plant)

Super division Spermatophyta (seed plants)

Division Magnoliophyta (flowering plants)

Class Magnoliopsida (Dicotyledone)

Sub class Asteridae

Order Solanales

Family Solanaceae

Genus Withania

Species Withania somnifera

FIG:ROOTS OF WITHANIA SOMINIFERA


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Chemical Constituents:-

The major biochemical constituents of ashwagandha roots are steroidal alkaloids and

steroidal lactones in a class of constituents called withanolides. At present, 12 alkaloids,

more than 35 withanolides, and several sitoindosides from this plant have been isolated

and studied. Much of ashwagandhas pharmacological activity has been attributed to its

various withanolides.

Active compound of withania somnifera

1. Withaferin A

2 .Diacetylwithaferin A

3. 2,3-Dihydrowithaferin A

4 .Withanolide viscosalactone B

5 .27-o-b-D-Glucopyranosyl viscosalactone B

6. Dihydrowithaferin A

7. 27-o-Glucopyranosylwithaferin A

8. Withanoside I-VII

9. 24,25-Dihydro-27-desoxywithaferin A

10. Physagulin D(16)-b-D-glucopyranosyl-(14)-b-D-glucopyranoside11. Sitoindosides VII-X

12.27- o-b-D-Glucopyranosylphysagulin D

13. 5-Dehydroxywithanolide R

14. Withasomniferin A

15. 1-Oxo-5b,6b-epoxy-witha-2-ene-27-ethoxy-olide

16. 4-(1-Hydroxy-2,2-dimethylcyclopropanone)-2,3-dihydrowithaferin A

17. D-Glucopyranosyl

18. D-Glucopyranoside

19. 4,16-Dihydroxy-5b,6b-epoxyphysagulin Dl


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Medicinal Use:-

InAyurveda, Ashwagandha is considered as a rasayana herb, which works on a nonspecific

basis to increase health and longevity. W. somnifera has been in use for over 2500 years to

treat all kind of diseases and human ailments (Bhattacharya A, et al., 2001). This herb is also

considered as an adaptogen which is a nontoxic herb that works on a nonspecific basis to

normalize physiological function, working on the HPA axis and the neuro-endocrine system.

The roots and berries of the plant are used in herbal medicine. In Ayurveda, the fresh roots are

sometimes boiled in milk, prior to drying, in order to leach out undesirable constituents. The

berries are used as a substitute for rennet, to coagulate milk in cheese making (Puri HS, 2003).

The species name somnifera means "sleep-bearing" in Latin, indicating it was considered a

sedative, but it has been also used for sexual vitalityand as an adaptogen. In the traditional

system of medicine Ayurveda, this plant is claimed to have potent aphrodisiacrejuvenative and

life prolonging properties. It has general animating and regenerative qualities and is used

among others for the treatment of nervous exhaustion, memory related conditions, insomnia,

tiredness potency issues, skin problems and coughing It improves learning ability and memory

capacity. The traditional use of Ashwagandha was to increase energy, youthful vigour,

endurance, strength, health, nurture the time elements of the body, increase vital fluids, muscle

fat, blood, lymph, semen and cell production. It also helps to counteract chronic fatigue,

weakness, dehydration, bone weakness, loose teeth, thirst, impotency, premature aging

emaciation, debility, convalescence and muscle tension. It helps to invigorate the body by

rejuvenating the reproductive organs, just as a tree is invigorated by feeding the roots

(Nadakarni.1993, Vaidyaratnam PS Variers.1994, 14. Sharma PV 1997). Fruits, leaves and

seeds of this plant have been traditionally used for the Ayurvedic system as aphrodisiacs,

diuretics and for treating memory loss. The Japanese patent applications are related to the use

of the herb as a skin ointment and for promoting reproductive fertility. In US, the NewEngland Deaconess Hospital, has taken a patent on an Ashwagandha formulation claimed to

alleviate symptoms associated with arthritis (Panda and Kar., 1997). The productcalled

"ashwagandha oil" is a combination of ashwagandha with almond oil and rose water designed


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to be used as a facial toner. Two acyl steryl glucosides, sitoindoside VII and sitoindoside

VIIIisolated from the roots of Withania somnifera were screened for putative antistress

activity using a diverse spectrum of stressinduced paradigms. Two new Glycowithanolides,

Sitoindoside IX (1) and Sitoindoside X (2) isolated from Withania somnifera were evaluatedfor their immunomodulatory and CNS effects (anti-stress, memory and learning) (3) It is said

to have free radical scavenger activity (antioxidant activity) in In vivo model where it has

increased superoxide dismutase and catalase activities of rat liver (Panda S and Kar A.1997).

The active principles of Withania somnifera consisting of equimolar amounts of Sitoindosides

VIIX and Withaferin A were investigated for putative nootropic activity in a experimentally

validated Alzheimers disease model. The syndrome was induced by ibotenic acid (IA)

lesioning of the nucleus basalis magnocellularis in rats. Withania somnifera significantly

reversed both IA induced cognitive deficit and the reduction in cholenergic markers after 2

weeks of treatment. These findings validate the Medharasayan (promoter of learning and

memory) effect of Withania somnifera as has been reported in Ayurveda (Bhattacharya SK, et

.al., 1994). Studies of ashwagandha showed that an acetone extract of alkaloids caused mild

CNS depression in dogs and mice and protected against supramaximal electroshock seizures

in rats. The extract caused hypothermia in mice and potentiated hypnosis induced by

barbiturates, ethanol and urethane (Malhotra CL, et.al., 1965) Root of Withania somnifera used

for the treatment of asthma, bronchitis, edema, leucoderma, anorexia, consumption, asthenia,

anemia, exhaustion, aging, Veena Sharma et al /Int.J. PharmTech Res.2011,3(1) 190

insomnia, ADD/ADHD, neurasthenia, infertility, impotence, repeated miscarriage, paralysis,

memory loss, multiple sclerosis, immune- dysfunction, carcinoma, rheumatism, arthritis,

lumbago (Nadkarni ,1976). Leaves have been used internally for fever and haemorrhoids;

externally for wounds, haemorrhoids, tumours, tuberculosis glands, anthrax pustules,

syphilitic sores, erysipelas, and in ophthalmitis (Kirtikar KR ,1953) Fruits are used externally

in ringworm (Varrier, 1996) A methanolic & 80% ethanolic extract of Withania somnifera

displayed significant anti-inflammatory activity on carrageenan- induced paw edema 2. The

root extract of Ashwagandha prevented the rise of experimentally induced LPO in rabbits &

mice (Dhuley JN.1998). Withaferin A and Sitoindosider VIII-X exhibits fairly potent anti-

arthritic, anti- inflammatory, antioxidant & immuno modulant activities, they also increase in


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the levels of SOD, CAT, GPX in brain & the steroidal lactone W.A ( Bhattacharya ,et.al.,

1997). Withaferin A, also showed significant antitumor & radiosensitizing effects in

experimental tumors without any toxicity & inhibiting tumor growth increasing survival in

swiss albino mice inoculated with Ehrlich ascites (ESC) carcinoma (Devi PU et.al.,1995) Theadministration of Ashwagandha Rasayana significantly reduced the lung tumor nodule

formation and also reduced leucopenia induced by cyclophosphamide treated experimental

animals, indicating its usefulness in cancer therapy (Menon LG,et.al.,1965). Withania increase

the WBC count, reduce leucopenia. They also increased bone marrow cellularity &

normalised the ratio of hormachromatic erythrocytes & polychromaticerythrocytes

Benefits:-

It is considered as the most important adaptogen in ayurvedic system of medicine. Itrelieves stress due to presence of vata suppressant properties which helps in nurturing

nervous system.

It works as a rasayan i.e. a substance that helps in preventing early aging andrejuvenates the whole body to provide youth. Its antioxidant properties help in

avoiding symptoms of early aging.

It increases muscular endurance and helps in building up of stamina. It works as a powerful immune booster that helps in fighting any foreign invasion in

the body. It is a wonderful remedy in increasing physical endurance in physically weak

people or people who are recovering from long illness as in case of tuberculosis or

surgeries.

It helps in promoting calmness and mental satisfaction in mind due to its goodpenetrating powers.

It improves mental ability, helps in gaining retaining power and improves mentalconcentration. It also helps in providing nourishment to the brain for its better function

and greater ability to work. It helps in promoting calmness and mental satisfaction in

mind due to its good penetrating powers.

It revitalizes body and decreases untimely fatigue caused due to weak body strength


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and accumulation of negative energies in the body.

It is often given to a person who regularly suffers from vertigo, incautiousness anddepression as it helps in curbing mental and physical weakness.

It is a powerful aphrodisiac, thereby enhancing the sexual powers and long lastingendurance. It also helps in increasing sperm count and the quality of sperm.

It is considered as one of the most commonly used herb in relieving hypertension withexcellent results.

It has also been found as an excellent supplement that helps in providing strength toheart muscles and keeps the heart working normal


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MATERIAL AND METHODS

3.1 Place of Work

The present work was carried out in Research Center, Mahavir Cancer Sansthan ,

Phhulwarisharif , Patna .

3.2 Experimental Laboratory animals

Experiments were done on Charles Foster rats .The animals were procured from the

animal house of Mahavir Cancer Sansthan and Research Center, Patna. There are several

reasons to select Charles Foster rats as the animal of choice for the present investigation.

These are as following:

1. Their Physiological activity is almost similar to that of humans (as 90 % of theirgenes are similar to humans).

2. Inbred strain.3. Small size.4. Early puberty (Sexual maturity).5. Short gestation period.6. High fecundity.7. Relatively high position in evolutionary science.

The weight of rats were ranging from 150225gm and 810 weeks of age.

3.2.1 Housing

Rats were kept separately in the ratio of two females per male in cages for

experimentation. Rats were kept in cages that are compatible with life, health, and

comfort, in such a way that regular needs of the animals, like feeding, watering, cleaning,

handling and the turnover of stock could be conveniently met.

In the present investigation polypropylene cages have been used. It had covers made of

stainless steel wires. Cages of size 40 x 25 x 15 (h) cm. were used..


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The dimensions of the cages were adequate to house a pair of rat and their litter till

weaning. Such cages could house three or four rats. These cages were also used for

mating purpose and stock purpose.

Rice husk were purchased from local market and were used for bedding after proper

sterilization. Husk bedding absorbs and provides comfort to the animal. It was changed

afresh every day and the used ones were thrown and properly disposed off. During the

treatment of Arsenic large cages were used, keeping male and female rats three in each

cage.

3.2.2 Physical Environment

The temperature of the rat experimentation room was in the range of 24C28C. Twelvehours of light and twelve hours of darkness were provided in the room for their optimal

growth and reproduction. The light intensity and humidity of the room was maintained at

an optimal level. The level of noise in the room was reduced to a minimum. Regular

cleaning of the room by using proper disinfectant (Lyzol) maintained pathogen free,

hygienic conditions of the rat room.

The facilities of electrical exhaust fan, cooler, other fans, cross ventilation through netted

windows etc.were made. All equipments of the various kinds used in the rat room were

disinfected. Dissecting sets like scissors, forceps, surgical blades, needles etc. were

washed thoroughly, sterilised and were kept in incubators at 80C. As recommended by

Templeton, (1945), the laboratory rats were fed on laboratory prepared enriched bread,

having the composition given below:


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Table 3.1: Food ingredients for laboratory rats per kg body weight:

In order to make the feed more enriched with vitamins, minerals etc, about 2 gm of green

vegetables such as spinach, carrot, sprouted grams were also given to each mouse. In

addition to all these, about 2 grams each per rat to make feed more enriched with

vitamins, minerals etc. The diet has 16% to 24% protein, 4% to 5 % fat, and 45% to 55%

carbohydrate. All the above-mentioned constituents were mixed properly and pellets of

equal size and weight were made manually. Weight of each feed pellet was

approximately 6 gm. In each cage one pellet of feed rat was given. The diet was palatable

to the animal as evidenced by feeding success. It has been observed that an adult rat

normally intakes 4 to 5 gm of diet per day. The daily food consumption of the rat varied

depending upon the physiological and health status of the rat as well as the environmental

temperature. The consumption of food increased considerably when the rat were pregnant

S.No. Ingredients Weight

1. Wheat grains 1 kg

2. Choker wheat 250 gm

3. Grams grains 250 gm

4. Maize grains 250 gm

5. Soybean grains 250 gm

6. Refined oil 50 gm

7. Milk powder 2 table spoon

8. Jaggery (Gudd) 50 gm.


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or at lactating stage and decreased considerably when the rat were provided with the

pesticide treatment depending upon dose-duration and increased temperature in summer.

Table : List of Instruments Used:-

Instruments Manufacturer

Incubator Acme instruments , India

Centifuge REMI, India

Colorimeter Photochem - micro , India

Microtome Amar Udyog , India

Hot Air Oven Acme instruments , India

Digital Camera Olympus- Japan

Microscope Olympus- Japan

Electronic Weighing Machine Setra BL - 3105 , India

UV Visible Spectrophotometer Beckman , USA

3.3 Experimental Protocol:-

The experiment was designed on day dependent basis.

Doses given on per kg body weight basis gave better results in rat.

3.4 Dosage of Cyclophosphamide:-

A drug marketed as Indoxan having composition of cyclophosphamide was made available by

MCS, Patna, Bihar, India .This drug is having 50mg of CPA and the total weight is 237mg . Rat

was supposed to administer 300mg CPA/kg body weight. Since 1000gm of Rats were supposed

to administer 300mg of CPA, hence, for 200gm of rats 60mg of CPA were administered. Since

237 mg of tablet contains 50mg of CPA, hence, for 60mg of CPA 284.4mg of tablet powder were

diluted in 2ml of distilled water and administered orally. Similarly the different doses were made

as per body weight.


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3.5 Preparation of Medicinal Extract:-

The medicinal plant used for the experiment was Withania somnifera (Ashwagandha).

Fresh root part of Withania species were made available by Mahavir Cancer Sansthan

and Ressearch Center, Patna, Bihar, India. It was washed in tap water and left for air dry.Then it was kept in incubater at 37-40

oc for 24 -48 hrs until it dries and crushed in mortar

and pistel to make it into powder form. Weight of the powder was taken and diluted for 5

times in 70% of alcohol and were left for 24 hrs to soak. Ethanolic extract was obtained

by using Sauxselet. Residual solid part was again dried and crushed to make fine powder.

Then it was filtered and 200 mg/kg body weight of W. somnifera wasdiluted in 5% of

ethanol and administerd to rats orally.

3.6 Rat grouping and Treatment of Rat:-

The rats were divided into the following groups first group were sacrificed after 45 days

of Sodium Arsenite treatment.The medicinal plant pleurotus cornicopie extracts treated

rat groups were sacrificed after 4 weeks of the treatment.

There were the follow ing 3 groups of Charles Foster Rat:

1. Normal control (n = 6)2. CPA (n = 6)3. W .Somnifera+ CPA (n = )

3.7 Heamatological study:-

3.7.1 Heamoglobin %

Haemoglobin % of normal, CPA treated, CPA and W. Somniferatreated rats respectivelywere analysed using heamometer .

20 l of N/10 HCl was taken in haemometer and 20 l of blood sample was taken and

mixed well. It was then diluted drop wise drop until the color of the solution matches the

color indicator of haemometer and the value is noted.


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3.7.2 RBC Count

RBC count of normal control, arsenic treated, untreated control, pleurotus treated and

pleurotus +arsenic treated rats were done by ocular puncture. During ocular puncture the

10l of blood was collected in a 2000l of RBC diluting fluid and mixed well. RBC

count was made using an improved Neubauers chamber taking a drop of above

preparation in it and calculation were done at 40x magnification.

3.7.3 WBC Count

WBC count of normal control, arsenic treated, untreated control, pleurotus treated and

pleurotus +arsenic treated rats were done by ocular puncture. During ocular puncture the

10l of blood was collected in a 200l of WBC diluting fluid and mixed well. WBCcount was made using an improved Neubauers chamber taking a drop of above

preparation in it and calculation were done at 40x magnification.

3.10 Histopathological Study:-

3.10.1 CollectionofTissues

After sacrificing, the rat liver and kidney tissues were dissected out and washed

thoroughly in normal saline (0.85 %) and fixed in formalin.

3.10.2 Fixationandembedding

Small pieces of liver and kidney tissues from the sacrificed rats were fixed by the

following procedure for subsequent histological studies under light microscope.

3.10.3 Tissue Processing

Tissues were fixed for 24 hrs. (Left in the fixative for longer periods when required, and

then washed overnight under running tap water). The tissues were dehydrated in 30%

alcohol (30ml absolute alcohol were taken and dissolved in 70ml distilled water), 50%



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alcohol (90ml absolute alcohol were taken and dissolved in 10ml distilled water) and

100% absolute alcohol for 15 minute. The tissues were cleaned in absolute alcohol with

xylene (1:1) and then in pure xylene for one hour. Then after passing through a mixture

of xylene and molten wax (1:1) for one hour, it was embedded in molten paraffin wax.

Blocks were made using L- moulds.

3.10.4 Sectioning

Blocks were fixed on the holder of the microtome.

Sections were cut at 5-6 thickness.

5-6 thick sections were fixed on Mayers albumin rubbed glass slides.

3.10.5 Staining

The sections were deparaffinised in xylene and hydrated through descending series 100%

absolute alcohol, 90% alcohol (90ml absolute alcohol were taken and dissolved in 10ml

distilled water), 70% alcohol (70ml absolute alcohol were taken and dissolved in 30ml

distilled water), 50% alcohol (50ml absolute alcohol were taken and dissolved in 50ml

distilled water) and then 30% alcohol (30ml absolute alcohol were taken and dissolved in

70ml distilled water). These hydrated sections were stained in haematoxyline for ten

minutes with one dip in acid water (if over stained). The sections were washed underrunning tap water at least for one hour and then rinsed in distilled water for

differentiation. Then the sections were dehydrated in ascending series 30% alcohol (30ml

absolute alcohol were taken and dissolved in 70 ml distilled water), 50% alcohol (50ml

absolute alcohol were taken and dissolved in 50ml distilled water), 70 % alcohol and

counter stained in eosin, (since eosin is prepared in 70% alcohol) dehydrated further in 90

% and absolute alcohol. Thereafter sections were clean in xylene and mounted in DPX

with clean glass cover slip. Slides were dried and then were viewed under compound

microscope.


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3.11. Statistical analysis:

Results were expressed as the mean value MEAN SD. Statistical differences between

groups were assessed by students ttest. Values of p 0.005 and p0.05 were considered

significantly different.

Arithmetic Mean:

Arithmetic mean x = x

n

Where,

x: Sum of the observations.

n: Number of observations.

Standard Deviation: This is used to measure the spread of the values in a series of

measurements. It is denoted by symbol S.D. and is calculated by the formula.

S.D. = (x1x) + (x2x) +. (xnx)

n or n -1

Where

x1, x2...xn= are individual valuesx = mean values

n = number of observations

If the number of observation is less than 30, factor n-1 is used.

Studentt test:

t = x1x2

(1/n1+ 1/n2) x SD2

Where,

x1= mean of 1stset


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x2= mean of 2nd

set

SD = 1 [(n11) SD1 + (n21) SD2]

(n1+ n22)

Results are presented as mean S.D and total variation present in a set of data was

analysed through one-way analysis of variance (ANOVA). Difference among means has

been analysed by applying Dunnet t test at 99.9% (p 0.001) confidence level.

Calculations were performed with the GraphPad Prism Program (GraphPad Software,

Inc., San Diego, USA).


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Objectives:-

1. To evaluate the effect of Cyclophosphamide on Charles foster rat throughhaematology, LFT and Histopathological study.

2. To evaluate the effect of W. somnifera on cyclophosphamide induced toxicitythrough haematology, LFT and Histopathological study.

3. To evaluate the antitoxic anti inflammatory and protective effect of W. Somniferaon Cyclophosphamide induced toxicity.

4. To establish the therapeutic dose of W. Somnifera as the antidote againstCyclophosphamide induced toxicity in rats


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Results:-

Table 4.1: Loss in body weight after 300 mg/ kg body weight CPA administration

and death rate of Charles Foster rats .

SEX

DAY

FIRST

DAY

THIRD

DAY

FIFTH

DAY

SEVENTH

DAY

TENTH

MALE-1 175 165 150 145 DEATH

MALE-2 200 195 180 170 DEATH

MALE-3 235 230 220 200 DEATH

Graph4.1: Graph showing loss in body weight of CPA administered Charles Foster rats

and its mortality effect .

235 230 220 200

0

200 195180

170

0

175165

150145

0

FIRST THIRD FIFTH SEVENTH TENTH

EFFECT OF CYCLOPHOSPHAMIDE (250mg/kg BODY

WEIGHT) ON MALE RAT

DEATH

DAYS


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Table 4.2: Comparison of RBC count among normal control, CPA treated &

Withania somnifera 200mg/kg body weight treated.

GROUPS RBC COUNT 106

/mm3

NC 5.265X10

CPA 2.940X10

WITHANIA 3.970 X10

NCP

A

WITHA

NIA

0

2.0106

4.0106

6.0106

1

06/mm

3

Graph 4.2:Graph shows that Withania somnifera administered orally dailyfor 10 days

has significantly elevated the level of RBC count in CPA treated Charles Foster rats in

which the RBC count has been decreased..


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Table 4.3: Comparison of RBC count among normal control, Withania somnifera

800mg/kg body weight treated & CPA300 mg/kg body weight treated Charles

Foster rats.

GROUPS RBC COUNT 10/mm

NC 5.100 X106

WITHANIA 7.312 X106

CPA 4.393 X10

6

NC

WITHA

NIA

CPA

0

2.0106

4.0106

6.0106

8.0106

106/m

m3

Graph 4.3:Graph shows that Withania somnifera 200mg/kg body weight administered

orally daily for 10 days and then administration of 300mg/kg body weight has

significantly inhibited the depletion in level of RBC count.


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Table 4.4: Comparison of WBC count among normal control, CPA 300mg /kg

bodyweight treated & Withani a somnifera 200mg/kg body weight treated.

GROUPS WBC COUNT 10/mm

NC 6850

CPA 2133

WITHANIA 4083

NC CPA

WITHA

NIA

0

2000

4000

6000

8000

103/mm

3

Graph 4.4:Graph shows that Withania somnifera administered orally dailyfor 10 days

has significantly elevated the level of WBC count in CPA treated Charles Foster rats in

which the WBC count has been decreased.


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Table 4.5: Comparison of WBC count among normal control, Withania somnifera

200mg/kg body weight treated & CPA 300mg/kg body weight treated Charles

Foster rats.

GROUPS WBC COUNT 10 /mm

NC 6850

WITHANIA 12400

CPA 7505

NC

WITHA

NIA

CPA

0

5000

10000

15000

103/mm

3

Graph 4.3:Graph shows that Withania somnifera 200mg/kg body weight administered

orally daily for 10 days and then administration of 300mg/kg body weight has

significantly inhibited the depletion in level of WBC count.


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Table 4.4: Comparison of serum Glutamic puruvate Transaminase level among

normal control, CPA 300 mg/ kg body weight & W.somnifera 800mg/kg body weight

treated Charles Foster rats.

GROUPS SGPT(U/ml)

NC 19.5

CPA 88.83

WITHANIA 65.83

NCCP

A

WITHA

NIA

0

20

40

60

80

100

U/ml

Graph 4.4: Graph shows that W.somnifera administered orally, daily for 10 days has

significantly decreased the serum SGPT level in Charles Foster rats which was elevated

due to 300mg/kg body weight CPA intoxication.


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Table 4.5: Comparison of serum Glutamic puruvate Transaminase level among

normal control, W.somnifera 800mg/kg body weight & CPA 300 mg/ kg body weight

treated Charles Foster rats.

GROUPS SGPT(U/ml)

NC 19.5

WITHANIA 15.0

CPA 47.83

NC

WITHA

NIA

CPA

0

20

40

60

U/ml

Graph 4.5:Graph shows that W.somnifera administered orally, daily for 10 days before

CPA 300mg/kg body weight intoxication has significantly inhibited the elevation of

serum SGPT level in Charles Foster rats


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GROSS APPEARANCE OF TESTIS IN DIFFERENT GROUPS OF RATS.

Fig. 4.1 Gross appearance of testes from all groups of rats. Illustrations of representativetestes in Control, CPA, W. somnifera and W. somnifera & CPA groups, respectively.

CPA group rats have obviously smaller testes as compared to the other three groups.


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HISTOPATHOOGICAL STUDY

PLATE - I

Photomicrographs of testicular sections of control (1), Cyclophosphamide (2),

Withania somnifera (3) and Cyclophosphamide +Withania somnifera (4) treated rats.

Testes from control (1) and Withania treated (3) rats exhibit a normal feature of

seminiferous epithelium and interstitial tissue with active spermatogenesis.

X200

However, a testis from a Cyclophosphamide treated rats (2) reveals markedly

atrophied seminiferous tubules with severe hypocellularity and impaired

spermatogenesis. Note Rupture, vacuolization, vascular congestion (black arrow),

oedematous fluid accumulation (white arrows) and interstitial space widening in

intertubular Connective tissue.

X200

Withania cotreated animals (4) display nearly normal architecture. Hematoxylin

and eosin

X200


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40/52



DISCUSSION:

Many drugs used for cancer chemotherapy are known to produce toxic side-effects in

multiple organ systems including the testes. In a clinical context, testicular stem cell

damage in patients exposed to chemotherapeutic drugs for a limited duration could result

in long-term infertility or genetic alterations (Sawada et al, 1994). A strategy to diminish

the side-effects of anticancer drugs with reservation of their chemotherapeutic efficacy is

necessary. Effective anticancer and immunosuppressive therapy with CP is severely

limited by testicular toxicity as documented in a variety of species (Anderson et al,

1995). An oxidant mechanism may be involved in the reproductive toxicity, wherein CP

and its metabolite acrolein cause inactivation of microsomal enzymes and result in

increased reactive oxygen species generation and lipid Peroxidation (Lear et al, 1992). Inthe present study, reduction in body weight, weight of the testis and epididymis and

histological changes in testis were indicative of drug toxicity. Because the weight of the

testis largely depends on the mass of the differentiated spermatogenic cells (Katoch et al,

2002), the marked reduction in organ weight by CP can be explained by diminished

number of germ cells, atrophy of Leydig cells and a significant lower rate of

spermatogenesis as confirmed by our findings. Reduction in the weight of testes and

epididymides in CP-treated animals reflect the reduced availability of androgens (Patil et

al 1998, ). Increased generation of free radicals is one of the possible mechanisms

involved in CP-induced Leydig cell degeneration resulted in marked reduction of serum

testosterone (Debnath et al, 2000). Chemotherapy can result in long-term or permanent

azoospermia, the mechanism of which is most likely the death of germ cells (Meistrich ,

1986) and stereological parameters such as seminiferous tubules diameters and their

epithelial heights, cross-sectional area of the seminiferous tubules, number of profiles of

seminiferous tubules in a unit area of testis and numerical density of seminiferous tubules

can also give information about the testicular damage degree as a consequence of germ

cell death. In general, massive germ cell loss caused by anticancer drugs is followed by a

sharp decline in testicular stereological parameters (Franka, et al, 1998). As shown in

present study, depletion of seminiferous epithelium and the consequent decrease of

morphometric and stereological measurements caused by cytotoxic agents were


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confirmed in our report. Structural development and maturation of germ cells and

spermiation are important functions of Sertoli cells (Mruk et al, 2004). Therefore, a

potential explanation for the failure of spermatogenesis in the CP-treated males is

disruption of testosterone- dependent junction of Sertoli cells with germ cells leading to

their disorganization and separation. In the present study, epididymal sperm count

decreased by, confirming a previous report that CP induces an epididymis specific effect

on sperm count (Higuchi et al, 2001). The decreased sperm count clearly shows the

elimination of sperm cells at different stages of development and points to free radical

attack through CP metabolism. In fact, oxidative damage to polyunsaturated fatty acids of

cell membranes has long been considered to result in the impairment of membrane

fluidity and permeability. This results in the damage of germ cells, spermatozoa and

mature sperm. It has also been reported that CP causes an increase in apoptosis at specific

stages of germinal cycle (Sikka, 2004). Hence, the decrease in epididymal sperm count

observed in CP-treated rats might reflect the spermatogenic cell death. There are several

reports on the benefit of antioxidants in protecting male reproductive system from

deleterious effects of reactive oxygen species and other free radicals generated during CP

exposure. It was found that ascorbic acid reduces cyclophosphamide-induced

reproductive toxicity as well as alpha-tocopherol-succinate. There is also evidence that

Yukmijihwang-tang as a multi-herbal medicinal formula can improve reproductive

toxicity of CP through reduction of oxidative stress (Oh, 2007). Two studies from the

same researchers indicated that supplementation with lipoic acid as an antioxidant

reduces CP-induced reproductive toxicity by the same mechanism. In the present study, it

has been shown that Withania somniferainflorescences aqueous extract coadministration

was effective in protection or attenuation of testicular damage following CP exposure.

Increasing evidences support the fact that Withania is beneficial where free radicals are

known to play a predominant role in toxicity. Previous studies have shown Withania

somnifera rat stomach against gastric ulcers induced by reactive oxygen species due to its

antioxidant properties (Potrich I et al , 2010). Furthermore, it has been revealed that W.

somnifera infusions reduce H2O2-induced oxidative damage in human erythrocytes and

leucocytes, which is consistent with their total flavonoid and phenol contents

(Konyalioghu & Karamenders, 2005). In conclusion, the finding of our study indicate


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that cyclophosphamide can adversely damage the testicular tissue through imposing

oxidative stress, while W. somnifera inflorescences aqueous extract coadministration

could effectively prevent these adverse effects by effective inhibition of oxidative

processes and efficient scavenging of free radicals.

CONCLUSION:

The study shows that W.somnifera may reduce the side effect of Cyclophosphamide if it

would be administered to the cancer patients before cyclophosphamide administration.

The admistration W.somniferaafter administration of cyclophosphamide may also reduce

its toxicity. Hence the study shows the antitoxic effect of W.somnifera against

cyclophosphamide intoxication.


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