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

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 1

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 et al., 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 et al., 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 Molecular weight Density Melting point Boiling point Log Kow Water solubility Vapor pressure

Information 261.1a 1.479 g/cm3b 49.5C to 53Ca 336C b 0.63a 40 g/L at 20Ca 4.45 105 mm Hg at 25oc

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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 lymphoma Cyclophosphamide 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 lymphoma Cyclophosphamide 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 myeloma Cyclophosphamide 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) Leukaemia Cyclophosphamide 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 lymphoma Cyclophosphamide is used alone or in combination regimens for the treatment of advanced mycosis fungoides, a form of cutaneous T-cell lymphoma. (f) Neuroblastoma Cyclophosphamide alone is used in the treatment of disseminated neuroblastoma. Combination chemotherapy that includes cyclophosphamide is also used for this neoplasm. (g) Cancer of the ovary Cyclophosphamide 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 platinumcontaining agent for the treatment of advanced (Stage III or IV) epithelial ovarian cancer. (h) Retinoblastoma Cyclophosphamide is used in combination therapy for the treatment of retinoblastoma (i) Cancer of the breast Cyclophosphamide 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, acut e 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 in inflammatory 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, and nephritic 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 hostmediated 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 1 by 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 antiCytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 9

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 of cyclophosphamide 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:Interaction 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 invitro 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 acroleintreated human fibroblasts and in peripheral blood 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 serum albumin has been described (Noort et al., 2002). The single-cell gel comet assay is used to detect single-strand breaks and other alkali-labilelesions in DNA exposed to cyclophosphamide

1.Withania sominifera (Ashwagandha) With Therapeutic Value

FIG:WITHANIA SOMINIFERA
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Ashwagandha or Indian winter cherry is 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 Sub kingdom Super division Division Class Sub class Order Family Genus Species Plantae(plants) Tracheaobionta (vascular plant) Spermatophyta (seed plants) Magnoliophyta (flowering plants) Magnoliopsida (Dicotyledone) Asteridae Solanales Solanaceae Withania 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-glucopyranoside 11. 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:In Ayurveda, 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 New England 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 evaluated for 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 antiarthritic, anti- inflammatory, antioxidant & immuno modulant activities, they also increase in
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 17

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) The administration 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. It relieves 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 and rejuvenates 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 good penetrating powers.

It improves mental ability, helps in gaining retaining power and improves mental concentration. 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 and depression as it helps in curbing mental and physical weakness.

It is a powerful aphrodisiac, thereby enhancing the sexual powers and long lasting endurance. 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 with excellent results.

It has also been found as an excellent supplement that helps in providing strength to heart 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 their genes 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 150 225gm and 8 10 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..
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 20

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. Twelve hours 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:

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 21

Table 3.1: Food ingredients for laboratory rats per kg body weight:
S.No. 1. 2. 3. 4. 5. 6. 7. 8. Ingredients Wheat grains Choker wheat Grams grains Maize grains Soybean grains Refined oil Milk powder Jaggery (Gudd) Weight 1 kg 250 gm 250 gm 250 gm 250 gm 50 gm 2 table spoon 50 gm.

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

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 22

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 Incubator Centifuge Colorimeter Microtome Hot Air Oven Digital Camera Microscope Electronic Weighing Machine UV Visible Spectrophotometer Manufacturer Acme instruments , India REMI, India Photochem - micro , India Amar Udyog , India Acme instruments , India Olympus- Japan Olympus- Japan Setra BL - 3105 , India 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. Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 23

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 37-40o c 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 was diluted 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. Somnifera treated rats respectively were 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.
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 24

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. WBC count 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 Collection of Tissues 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 Fixation and embedding 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% alcohol (50ml absolute alcohol were taken and dissolved in 50ml distilled water), 70%
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 25

alcohol (70ml absolute alcohol were taken and dissolved in 30ml distilled water), 90% 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 under running 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.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 26

3.11. Statistical analysis: Results were expressed as the mean value MEAN SD. Statistical differences between groups were assessed by students t test. Values of p 0.005 and p0.05 were considered significantly different.
Arithmetic Mean:

Arithmetic mean x = Where,

x n

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. =

(x1 x) + (x2 x) +. (xn x)

n or n -1

Where x1, x2...xn = are individual values x = mean values n = number of observations

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

Studentt test: t =

(1/n1 + 1/n2)
Where, x1 = mean of 1st set

x1 x2

x SD2

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 27

x2 = mean of 2nd set SD = 1 [(n1 1) SD1 + (n2 1) SD2] (n1 + n2 2)

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).

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 28

Objectives:1. To evaluate the effect of Cyclophosphamide on Charles foster rat through haematology, LFT and Histopathological study. 2. To evaluate the effect of W. somnifera on cyclophosphamide induced toxicity through haematology, LFT and Histopathological study. 3. To evaluate the antitoxic anti inflammatory and protective effect of W. Somnifera on Cyclophosphamide induced toxicity. 4. To establish the therapeutic dose of W. Somnifera as the antidote against

Cyclophosphamide induced toxicity in rats

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 29

Results:Table 4.1: Loss in body weight after 300 mg/ kg body weight CPA administration and death rate of Charles Foster rats .

DAY SEX FIRST

DAY THIRD

DAY FIFTH

DAY SEVENTH

DAY TENTH

MALE-1 MALE-2 MALE-3

175 200 235

165 195 230

150 180 220

145 170 200

DEATH DEATH DEATH

EFFECT OF CYCLOPHOSPHAMIDE (250mg/kg BODY WEIGHT) ON MALE RAT

175 200 165 195 150 180 220 145 170 200

235

230

DEATH
FIRST THIRD FIFTH SEVENTH 0 TENTH

DAYS

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

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 30

Table 4.2: Comparison of RBC count among normal control, CPA treated & Withania somnifera 200mg/kg body weight treated.

GROUPS

RBC COUNT 106 /mm3 5.265X106 2.940X106 3.970 X106

NC CPA WITHANIA

6.010 6

106/mm3

4.010 6

2.010 6

PA

Graph 4.2: Graph shows that Withania somnifera administered orally daily for 10 days has significantly elevated the level of RBC count in CPA treated Charles Foster rats in which the RBC count has been decreased..

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 31

IT H A

IA

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 106 /mm3

NC WITHANIA CPA

5.100 X106 7.312 X106 4.393 X106

8.010 6 6.010 6

106/mm3

4.010 6 2.010 6 0

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.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 32

W IT

H A

P A

IA

Table 4.4: Comparison of WBC count among normal control, CPA 300mg /kg bodyweight treated & Withania somnifera 200mg/kg body weight treated.

GROUPS

WBC COUNT 103 /mm3

NC CPA WITHANIA

6850 2133 4083

8000 6000

103/mm3

4000 2000 0

P A

Graph 4.4: Graph shows that Withania somnifera administered orally daily for 10 days has significantly elevated the level of WBC count in CPA treated Charles Foster rats in which the WBC count has been decreased.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 33

IT

H A

IA

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 103/mm3

NC WITHANIA CPA

6850 12400 7505

15000

103/mm3

10000

5000

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.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 34

IT

H A

P A

IA

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 CPA WITHANIA

19.5 88.83 65.83

100 80

U/ml

60 40 20 0

P A

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.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 35

W IT

H A

IA

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 WITHANIA CPA

19.5 15.0 47.83

60

U/ml

40

20

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

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 36

IT

H A

P A

IA

GROSS APPEARANCE OF TESTIS IN DIFFERENT GROUPS OF RATS.

Fig. 4.1 Gross appearance of testes from all groups of rats. Illustrations of representative testes 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.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 37

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

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 38

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 39

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). In the 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
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 40

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 somnifera inflorescences 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
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 41

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.somnifera after administration of cyclophosphamide may also reduce its toxicity. Hence the study shows the antitoxic effect of W.somnifera against cyclophosphamide intoxication.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 42

Reference:
Akron. 2009. The Chemical Database. The Department of Chemistry at the University of Akron. http://ull.chemistry.uakron.edu/erd and search on CAS number. Last accessed: 11/14/09.

Chabner BA, Ryan DP, Paz-Ares L, Garcia-Carbonero R, Calabresi P. 2001. Antineoplastic agents. In Goodman & Gilmans The Pharmacological Basis of Therapeutics, 10th ed. Hardman JG, Limbird LE, eds. New York: McGraw Hill. pp. 1389-1459.

ChemIDplus. 2009. ChemIDplus Advanced. National Library of Medicine. http://chem.sis.nlm.nih.gov/chemidplus/chemidheavy.jsp and select Registry

Number and search on CAS number. Last accessed: 11/14/09 . ChemSources. 2009. Chem Sources - Chemical Search. Chemical Sources International. http://www.chemsources.com/chemonline.html and search on cyclophosphamide. Last accessed: 10/22/09.

ClinicalTrials. 2009. Cyclophosphamide. ClinicalTrials.gov. National Institutes of Health. 10/29/09 . Evelo CT, Bos RP, Peters JG, Henderson PT. 1986. Urinary cyclophosphamide assay as a method for biological monitoring of occupational exposure to cyclophosphamide. Int Arch Occup Environ Health 58(2): 151-155. http://clinicaltrials.gov/ct2/results?term=azacitidine. Last accessed:

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 43

 FDA. 2009. The Electronic Orange Book. U.S. Food and Drug Administration. http://www.fda.gov/cder/ob/default.htm and select Search by Active Ingredient and search on cyclophosphamide. Last accessed: 10/26/09.

HSDB. 2009. Hazardous Substances Data Bank. National Library of Medicine. http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB and search on CAS number. Last accessed: 10/22/09

IARC. 1975. Cyclophosphamide. In Some Aziridines, N-, S-, and O-Mustards and Selenium. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, vol. 9. Lyon, France: International Agency for Research on Cancer. pp. 135-156.

IARC. 1981. Cyclophospamide. In Some Antineoplastic and Immunosuppressive Agents. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, vol. 26. Lyon, France: International Agency for Research on Cancer. pp. 165-202.

IARC. 1987. Cyclophosphamide. In Overall Evaluations of Carcinogenicity. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, suppl. 7. Lyon, France: International Agency for Research on Cancer. pp. 182-184.

MedlinePlus.

2009.

Cyclophosphamide.

National

Library

of

Medicine. Last

http://www.nlm.nih.gov/medlineplus/druginfo/meds/a682080.html. accessed:1/7/10.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 44

 NIOSH. 1990. National Occupational Exposure Survey (1981-83). National Institute for Occupational Safety and Health. Last updated: 7/1/90.

http://www.cdc.gov/noes/noes1/x3688sic.html.

RxList.

2010.

Cytoxan.

RxList:

The

Internet

Drug

Index.

http://www.rxlist.com/cytoxan-drug.htm. Last accessed: 1/7/10.

Travis LB, Curtis RE, Glimelius B, Holowaty EJ, Van Leeuwen FE, Lynch CF, et al. 1995. Bladder and kidney cancer following cyclophosphamide therapy for non-Hodgkins lymphoma. J Natl Cancer Inst 87(7): 524-530.

Zimmerman PF, Larsen RK, Barkley EW, Gallelli JF. 1981. Recommendations for the safe handling of injectable antineoplastic drug products. Am J Hosp Pharm 38(11): 1693-1695.

Isselbacher.Braunwald.Wilson.Martin Fauri Kasper. HarrisonsPrinciples Of Internal Medicine, 13th edition: McGraw-Hill,Inc ; 1994. p.1829 Isselbacher.Braunwald.Wilson.Martin Fauri Kasper .HarrisonsPrinciples Of Internal Medicine, 13th edition : McGraw-Hill,Inc; 1994.p. 1834 Leemol Davis. Girija Kuttan.Suppressive effect of cyclophosphamide-induced toxicity by Withania somnifera extract in mice, Journal of Ethnopharmacology;62. p. 209214 .Dorlands. Illustrated Medical Dictionary, 30th edition: Saunders An Imprint Of Elsevier; 2000.p. 470-471

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 45

 . Dr Brahmanand Tripati. Astanga Hridayam Of Sri Madhava Bhata: Chaukamba Sanskrit Pratishthan Delhi; 1999 .35th chap.p. 1142

Dr Brahmanand Tripati. Astanga Hridayam Of Sri Madhava Bhata: Chaukamba Sanskrit Pratishthan Delhi; 1999. 5th chap.p. 73 Leemol Davis. Girija Kuttan.Suppressive effect of cyclophosphamide-induced toxicity by Withania somnifera extract in mice, Journal of Ethnopharmacology;62. p. 209214

Acharya Priyavath Sharma.Dr Guru Prasad Sharma. Kaiyyadeva Nigantu Patyaapatya Vibhodaka: Chaukamba Oriyantalia Varanasi; 2009.p. 367 Dr Brahmanand Tripati.Sharangadhara samhitha of PanditaSarangadharacharya: Chaukamba Surbharati Prakashan;2004.p.218-220 Bristol-Myers Squibb Co. Cytoxan Package insert. Bristol-Myers Squibb Company. Princeton NJ. (2003) Chabner, B.A., D.P. Ryand, Pax-Ares L, Garcis-Carbonero and Calaresi, P. (2001) Antineoplastic Agents. In Goodman and Gilmans The Pharmacological Basis of Therapeutics, 10th ed. J.G. Harnam and L.E. Libmirde, eds NewYork, NY: McGraw Hill. 1389-1459 Chabner, B.A.et al. (2006) Antineoplastic Agents. In Goodman and Gilmans The Pharmacological Basis of Therapeutics, 11th ed. J Brunton LL, Lazo JS, and Parker KL, eds NewYork, NY: McGraw Hill. 1322-1328, 1694

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 46

 Gilian SH and Chatzinoff M (1983) Embryopathic effects of Cyclophosphamide. Environ Res 31:296-301 Greene, M.H., Harris, E.L., Gershenson, D.M., Malkasian, G.D., Jr, Melton, L.J., III, Dembo, A.J., Bennett, J.M., Moloney, W.C. & Boice, J.D., Jr (1986) Melphalan may be a more potent leukemogen than cyclophosphamide. Ann. int. Med., 105, 360-367

Hansel S, Castegnaro M, Sportouch MH, De Meo M, Milhavet JC, Laget M and Dumenil G. Chemical degradation of wastes of antineoplastic agents: cyclophosphamide, ifosfamide and melphalan. Int Arch Occup Environ Health. 1997; 69:109-114. Haas, J.F., Kittelmann, B., Mehnert, W.H., Staneczek, W., Mohner, M., Kaldor, J.M. & Day, N.E. (1987) Risk of leukaemia in ovarian tumour and breast cancer patients following treatment by cyclophosphamide. Br. J. Cancer, 55, 213-218

IARC (1981) Some Antineoplastic and Immunosuppressive Agents. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Vol 26. Lyon, France: International Agency for Research of Cancer. pp 411 IARC (1987) Overall Evaluation of Carcinogenicity. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Supplement 7. Lyon, France: International Agency for Research of Cancer. pp 440 Kinlen, L.J. (1985) Incidence of cancer in rheumatoid arthritis and other disorders after immunosuppressive treatment. Am. J. Med., 78 (Suppl. 1A), 44-49

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 47

 Masta, A., Gray, P. J., Phillips, D. R. (1995). Nitrogen mustard inhibits transcription and translation in a cell free system. Nucleic. Acids Res. 23:3508 3515.

Meirow D, Epstein M, Lewis H, Negent D, and Gosden RG (2001) Administration of Cyclophosphamide at different stages of follicular maturation in mice: effects on reproductive performance and feta malformations. Human Reproduction 16:632-637 NIOSH Alert (2004) Preventing Occupational Exposures to Antineoplastic and Other Hazardous Drugs in Health Care Settings. National Institute of Occupational Safety and Health. NIOSH-Publications Disseminations: Cincinnati, OH National Toxicology Program. Cyclophosphamide CAS No. 50-18-0 National Institute of Environmental Health Sciences. 11th Ed Report on Carcin. (2005)

OConnor, P. M., Wassermann, K., Sarang, M., Magrath, I., Bohr, V. A., Kohn, K. W. (1991). Relationship between DNA cross-links, cell cycle, and apoptosis in Burkitts lymphoma cell lines differing in sensitivity to nitrogen mustard. Cancer Res 51:65506557. Padmanabhan R, Singh S (1984) Congenital anomalies of the ear resulting from cyclophosphamide treatment in the rat. Acta Anat 119:217-223

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 48

 Pette M, Gold P, Pette DF, Hartung HP, and Toyka KV (1995) Mafosphamide induces DNA fragmentation and apoptosis in human T-lymphocytes. A possible mechanism of its immunosuppressive action. Immunopharmacology 30:59-69

Sessink PJM, Van der Kerkhof MCA, Anzion RBM, Noordhoek J, Bos RP (1993). Environmental contamination and assessment of exposure to

antineoplastic agents by determination of cyclophosphamide in urine of exposed pharmacy technicians: is skin absorption an important exposure route? Arch Environ Health 49(3):165169. Springer, J. B., Colvin, M. E., Colvin, O. M., Ludeman, S. M. (1998). Isophosphoramide mustard and its mechanism of bisalkylation. J. Org. Chem. 63:72187222. Stahlmann R, Bluth U, Neubert D (1985) Effects of cyclophosphamide metabolite acrolein in mammalian limb bud cultures. Arch Toxicol 57:163-167

Vaux KK, Kahole NCO, Jones KL (2003) Cyclophosphamide, Methotrezate, and Cytarabine Embropathy: Is Apoptosis the Common Pathway? Birth Defects Research 67:403-408 Allison P. Global survey of marine and estuarine species used for traditional medicine and tonic foods. WHO Report, McGill University, Quebee, Canada. 1966. Anonymous. Medicinal plants: Their Biodiversity, Screening and Evaluation. Center for Science and Technology of the Non- aligned and other developing countries, New Delhi.1998

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 49

 Bhattacharjee SK. Handbook of medicinal plants. Pointer publishers, Jaipur, India. 1998. Pattipati S, Amanpreet S, Shrinivas K. Effect of Withania somnifera root extract on Haloperidol induced Orofacial Dyskinesia: Possible mechanism of action. J. Med. Food 2003; 6(2): 107-114. Jayaprakasam B, Zhang Y, Seeram N and Nai M. Growth inhibition of human tumour cell lines by withanolides from Withania somnifera leaves. Life Sciences. 2003; 74: 125-132. Prakash J, Gupta S and Dinda A. Withania somnifera root extract prevents DMBA-inducedsquamous cell carcinoma of skin in Swiss albino mice. Nutrition cancer. 2002; 1: 91-97 Weiner MA and Weiner J. Ashwagandha (India ginseng). In: Herbs that. Quantum Book, Mill Valley, CA. 1994; 70-72. Sharma S, Dahanukar and Karandikar SM. Effects of long-term administration of the roots of ashwagandha and shatavari in rats. Indian Drugs. 1985; 29:133-139. Kokate CK, Purohit AP and Gokhale SB. Pharmacognosy, 4th edition, 624-629, Nirali Prakashan, Pune. 1996. Bhattacharya A, Ghosal S and Bhattacharya S. Antioxidant effect of WS glycowithanolides in chronic foot shock induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. Journal Ethnopharmacol. 2001; 74: 1 Puri HS. RASAYANA: Ayurvedic Herbs of Rejuvenation and Longevity. Taylor & Francis,
Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 50

 London. 2003; p. 46-58.12. N adakarni. Indian Materia Medica. 1993; 1:1292. 13. Vaidyaratnam PS Variers. Indian MedicinalPl ants, a compendium of 500 species. 1994. Sharma PV (1997) Dravyaguna Vigyan, Chowkambha Sanskrit Sansthan.15. Charaka Samhita and Chikitsa Sthana. Second Chapter, Chowkambha Publishers, 38 (EnglishEdition). 1997. Panda S and Kar A. "Evidence for free radical scavenging activity of Ashwagandha root powder in mice"; Ind. J. Physiol. Pharmacol.1997; 41(4): 424426. Bhattacharya SK, Kumar A and Ghosal S."Effects of Glycowithanolides form Withania somnifera on an animal model of Alzheimer's Disease and perturbed Central Cholinergic Markers of Cognition in rats"; Phytotherapy Research. 1994; 8: 1-4. Malhotra CL, Mehta VL, Das PK and DhallaNS. "Studies on Withania ashwagandha kaul (Part-V): The effect of total alkaloids(ashwagandholine) on the CNS, Ind. J. Physiol. Pharmacol. 1965; 127-136. Nadkarni and Dr. KM. The Indian Materia Medica, with Ayurvedic, Unani and Home Remedies. Revised and enlarged by A.K. 1976. 20. Frawley, David and Vasant Lad. The yoga of Herbs: An Ayurvedic Guide to Herbal Medicine Santa Fe: Lotus Press. 1986. Dash and Bhagwan. Materia Medica ofAyurneda. New Delhi: B.Jain Publishers. 1991.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 51

 Kirtikar KR and Basu BD Indian Medicinal plants. 2nd ed. Vol.1-4. 1935. Reprint. Delhi:Periodical Experts. 1993. Varrier. Mention that a paste made of the roots and bruised leaves may be applied to carbuncles,ulcers and painful swellings. 1996; p. 409. Hindawi -al MKIH, AI-Deen, Nabi MH and Ismail MH. Anti-inflammatory activity of some Iraqi plants using intact rats. Journal Ethnopharmacol. 1989; 26 (2): 1638. Dhuley JN. Therapeutic efficacy of Ashwagandha against experimental aspergillosis in mice. Immuopharmacol Immunotoxicol. 1998; 20 (1): 191-8. Bhattacharya SK, Satyan KS and Ghosal S. Antioxidant activity of glycowithanolides from Withania somnifera. Indian J Exp Biol. 1997; 35(3): 2369. Devi PU, Sharada AC and Solomon FE. In vivo growth inhibitory and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma. Cancer Lett. 1995; 16(1-2): 189-93. Devi PU. Withania somnifera Dunal (Ashwagandha): potential plant source of a promising drug for cancer chemotherapy and radiosensitization. Indian Journal Exp Biol.1996; 34 (10): 927-32. . Sharad AC, Solomon FE, Devi PU, Udupa N and Srinivasan KK. Antitumor and radiosenitizing effects of withaferin A on mouse Ehrlich ascites carcinoma in vivo. Acta Oncol. 1996; 35(1): 95-100.

Cytoprotective effect of Withania somnifera against cyclophosphamide induced toxicity in Charles Foster rats . Page 52