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Phytomedicine 12 (2005) 285–293

www.elsevier.de/phymed

Modulatory influence of Adhatoda vasica Nees leaf extract against gamma

irradiation in Swiss albino mice

A. Kumar�, J. Ram, R.M. Samarth, M. Kumar

Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur, India

Received 15 February 2003; accepted 3 December 2003

Abstract

The radiomodulatory influence of ethanolic extract of Adhatoda vasica Nees leaf extract against radiation-inducedhematological alterations in peripheral blood of Swiss albino mice was studied at various post-irradiation intervalsbetween 6 h to 30 days. Oral administration of A. vasica leaf extract (800mg/kg body weight) prior to whole bodyirradiation showed a significant protection in terms of survival percentage and hematological parameters. Miceexposed to radiation (8.0Gy) without A. vasica leaf extract pre-treatment exhibited signs of radiation sickness likeanorexia, lethargicity, ruffled hairs and diarrhoea and such animals died within 25 days post-irradiation. The dosereduction factor (DRF ¼ 1.6) for A. vasica leaf extract was calculated from LD50/30 values. A significant decline inhematological constituents (RBCs, WBCs, Hb and Hct) was evident till day 15 and no animal could survive beyondday 25. Conversely, animals pre-treated with A. vasica leaf extract showed 81.25% survival till 30 days after exposureand a gradual recovery was noted in the hematological values. However, these hematological values remainedsignificantly below the normal even till day 30. A significant decrease in blood reduced glutathione (GSH) content andincrease in lipid peroxidation (LPO) level was observed in control animals (Radiation alone). However, A. vasica leafextract pretreated irradiated animals exhibited a significant increase in GSH content and decrease in LPO level. Asignificant increase in the serum alkaline phosphatase activity and decrease in acid phosphatase activity was observedin A. vasica leaf extract pretreated irradiated animals during the entire period of study.r 2004 Elsevier GmbH. All rights reserved.

Keywords: Radioprotection; Gamma radiation; Hematological constituents; Adhatoda vasica; Survivality; GSH; LPO; Serum

phosphatases; Swiss albino mice

Introduction

Radiation-induced hematological alterations havebeen extensively studied. After whole body exposure,manifestations of injury to mammalian tissues are wellreflected in peripheral blood (Rugh and Somogyi, 1968;Samarth et al., 2001). The quantitative effects of whole-

e front matter r 2004 Elsevier GmbH. All rights reserved.

ymed.2003.12.006

ing author. Tel.: +91141 2711158;

01137.

ess: mamsjpr@hotmail.com (A. Kumar).

body irradiation are more significant than qualitativechanges in circulating cells (Block, 1976).Recently, interest has been generated to develop the

potential drugs of plant origin for the modification ofradiation effects. Plant extract such as garlic (Gupta,1988), ginseng (Pande et al.,1998), Aloe vera (Pande etal.1998), podophyllum (Goel et al., 1999), ocimum(Uma Devi et al., 2000), spirulina (Kumar et al.,2002), mentha (Samarth et al., 2002) have been foundto have radioprotective effects in mammals. Plantproducts appear to have an advantage over the synthetic

ARTICLE IN PRESSA. Kumar et al. / Phytomedicine 12 (2005) 285–293286

compounds in terms of low/no toxicity at the effectivedose with minimum/no side effects. However, the use ofmedicinal plants in modern medicine suffers from thefact that though several plant plants are used in theWorld to prevent or to cure diseases, scientific evidenceis lacking in most cases. Therefore, it is necessary toprovide scientific background as to whether it is justifiedto use a plant or its active principles (Ammon and Wahl,1991)

Adhatoda vasica, an evergreen, gregarious, stiff andperennial shrub of family Acanthaceae has been used asherbal medicine in treating a wide variety of diseases inIndia. Leaves of the plant are the main source of drugpreparation. A number of different principles including,alkaloids: vasicine, vasicinone, vasinol, essential oil:betane, vitamins: vitamin C, b-carotene, a non-crystal-line steroid: vasakin and a mixture of fatty acids havebeen identified as contributing to the observed medicinaleffects of the plant (Wealth of India, 1989). The shrub isthe source of the drug ‘vasaka’, well known in theindigenous system of medicine for its beneficial effects,particularly in bronchitis. Furthermore, A. vasica hasalso been accredited to afford protection againstallergen-induced bronchial obstruction in guinea pigs(Dorsch and Wagner, 1991). The leaves as well asflowers, fruits and roots are extensively used for treatingcold, whooping cough, asthma and as antihelmintic. Theleaf juice is stated to cure diarrhoea, dysentry andglandular tumor. In vivo investigations have revealedthat 90% ethanolic extract of A. vasica possesses apromising teratologic effects in rats (Nath et al., 1992).Bromohexine and ambroxol, semisynthetic derivativesof vasicine from A. vasica have growth inhibitory effecton Mycobacterium tuberculosis thereby proving useful inthe therapy of tuberculosis (Grange and Snell, 1996).One of the principal mechanisms of the radiation

damage is the production of free radicals and thescavenging of these free radicals by conjugation Phase IIenzymes results in radioprotection. Singh et al. (2000)showed that hydro alcoholic extract of A. vasica

modulate the Phases I and II enzyme system and thusresult in cancer chemoprevention in mice model system.Therefore, the present study has been undertaken toinvestigate the radioprotective activity of A. vasica

leaf extract in terms of body-weight, survivability,hematological and biochemical parameters of Swissalbino mice.

Materials and methods

Animals

Male Swiss albino mice, 6–8 weeks old with 2572 gbody weight, from an inbred colony (obtained from

Hamadard University, Delhi) were used for the presentstudy. Animals were maintained under controlledconditions of temperature and light in an animal house,and were provided standard mice feed (Procured fromHindustan Lever’s Ltd., Delhi) and water ad libitum.

Irradiation

Cobalt teletherapy unit (ATC-C9) at the CancerTreatment Centre, Radiotherapy Department, SMSMedical College & Hospital, Jaipur was used forirradiation. Unanaesthetized animals were restrainedin well-ventilated Perspex boxes and exposed whole-body to gamma radiation (with the source to surfacedistance (SSD) of 77.5 cm and 1.02Gy/min of dose-rate).

Adhatoda vasica leaf extract (AE)

A voucher specimen of A. vasica has been preserved inthe herbarium of Botany Department, university ofRajasthan, Jaipur (RUBL- 18822). A. vasica plants werecollected from Khetri, Rajasthan (India), their leaveswere washed thoroughly and shade dried; a knownquantity of the material was soxhleted using 80%ethanolic solvent (three changes). Finally, the extractwas lyophilized, weighed and preserved at 4 1C and usedas and when required.

Experimental design

Determination of optimum dose of Adhatoda vasica leaf

extract against radiation

Mice were divided into six groups of 10 animals eachand were given A. vasica leaf extract orally (200, 400,800, 1000 and 1200mg/kg body weight/day) for 7consecutive days. Thirty minutes after the last admin-istration, animals were exposed whole body to 8.0Gygamma radiation. All such animals were observed till 30days for any sign of radiation sickness, morbidity,behavioral toxicity and mortality. The optimum dosethus obtained was used for further investigation.

Survival assay

Mice both control and experimental, exposed wholebody to gamma radiation (6, 8 and 10Gy) were checkeddaily for 30 days. The survival percentage of mice up to30 days of exposure against each radiation dose wasused to construct survival dose–response curves. Re-gression analysis was done to obtain LD50/30 values andto determine dose reduction factor (DRF).

Modification of radiation response

Animals selected for this study were divided into twogroups. Animals of one group were administered A.

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vasica leaf extract orally (800mg/kg body weight/day)for 7 consecutive days to serve as an experimental whilethe other group received DDW (volume equal to A.

vasica leaf extract) to serve as control. After 30min ofabove treatments on 7th day, animals of both the groupswere exposed to gamma radiation (8.0Gy).

Body-weight

The general condition of the mice was observeddaily and recorded through the measurement of thebody weight. The per cent change in body weight foreach of the groups of mice was recorded daily bydividing the average weight of mice surviving on givenday by the average weight of the same mice treated onthe first day.

Hematological study

Blood was collected from tail vein in a vial containing0.5M EDTA. Peripheral blood counts (RBC/WBC),hemoglobin (Hb) concentration and hematocrit (Hct)percentage were determined at 6, 24 h and 15 and 30days post-irradiation by adopting standard procedures.

Biochemical study

Reduced glutathione (GSH) assay

The GSH content in blood was measured Spectro-photometrically using Ellman’s reagent (DTNB) as acoloring reagent as per the method described by Beutleret al. (1963). The absorbance was read at 412 nm using aUV-VIS Systronics Spectrophotometer.

Lipid peroxidation (LPO) assay

The LPO level in serum was measured in terms ofThiobarbituric Acid Reactive Substances [TBARS] bythe method of Ohkhawa et al. (1979). The absorbancewas read at 532 nm.

Serum phosphatases activity

Serum alkaline phosphatase activity was measured byKind and King’s method. Acid phosphatase activity wasdetermined by King’s method by using commerciallyavailable kits (Varley, 1980).

Statistical analysis

The results obtained were expressed as mean7SE.Student’s ‘t’-test was used to make a statisticalcomparison between the groups. Significance levels wereset at po0:05; po0:005 and po0:001: Regressionanalysis was done to obtain LD50/30 values and todetermine DRF.

Results

Animals treated with different concentration (200,400, 800, 1000 and 1200mg/kg body weight/day) for 7consecutive days of A. vasica extract (AE) showed noadverse effects such as sickness body weight change,mortality and visible abnormality during 30 days ofexperimentation. Optimum dose of AE against lethalgamma radiation (8.0Gy) in Swiss albino mice wasselected on the basis of survivability during experimen-tation. Mice treated with AE at the concentrations of800, 1000 and 1200mg/kg body weight/day for 7consecutive days prior to irradiation exhibited 81.25%,75.00% and 60.75% survivability respectively (Fig. 1).Animals treated with 800mg/kg body weight/dayshowed maximum survivability, therefore, 800mg/kgbody weight/day dose of AE was used for furtherdetailed study. The percentage of survival was signifi-cantly increased in AE treated irradiated animals.When survival data were fit on regression lineequation, the LD50/30 values for control and experi-mental group of animals were found as 6.4270.16 and10.3170.8Gy, respectively. These data were used forthe calculation of DRF and it was calculated as 1.6(Figs. 2 and 3).Animals exposed to 8.0Gy radiation showed signs

and symptoms of radiation sickness such as nausea anddiarrhoea and these animals became lethargic resultingin reduced food and water intake. No animal survivedtill day 30 post-irradiation in control (Radiation alone)group. These animals showed loss of weight up to31.32% from initial weight on day 25 post-irradiation(Fig. 4).Total RBC count was observed significantly below the

normal value, as early as at 6 h (4.8570.27� 106mm3,po0:005) interval. The maximum decline in theirnumber was noted at 24 h post irradiation(4.7670.09� 106/mm3, po0:005). An increase in num-ber of such cells was noted on day 15 (5.2470.05� 106/mm3, po0:005). However, the number of RBC couldnot increase up to normal value (9.4870.38) till day 30post-irradiation at 8Gy gamma radiation. WBC countdeclined significantly, on 24 h (3.8570.05� 103/mm3,po0:005) post irradiation. Gradual increase was noticedon day 15 (3.9770.06� 103/mm3, po0:005) post-irradiation. The number of WBC could not reach tonormal value (6.1870.36� 103/mm3) till day 30 postirradiation. Hemoglobin level declined significantly incomparison to normal (15.3270.22 gm/100ml). Max-imum declined was noted on day 15 (8.8270.11 gm/100ml, po0:005). The value of hemoglobin showedcontinuous decrease from 6h (9.9570.11 gm/100mlpo0:005), interval after exposure to 8Gy gamma. Thehematocrit percentage also exhibited a declining trendup to day 15 post-irradiation. A sharp decline inhemotocrit percentage was noticed. Hematocrit value

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Fig. 2. 30 Days survival of mice, pretreated with or without AE (800mg/kg b.wt.) for 7 consecutive days and exposed to different

doses of gamma radiation.

Fig. 1. 30 Days survival of mice, pretreated with different doses of AE for 7 consecutive days and exposed to 8Gy gamma

radiations.

Fig. 3. Survival–dose–response curves for determination of LD50/30.

A. Kumar et al. / Phytomedicine 12 (2005) 285–293288

gradually decreased from 6h (30.370.38%; po0:005) to15 days (24.3570.33%; po0:005) of autopsy intervals(Table 1).

In AE treated and irradiated animals, maximumdecline in total RBC count (6.9670.08� 106/mm3,po0:005) was observed at 6 h autopsy interval. The

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Fig. 4. Body weight response of mice pretreated with or without AE and exposed to different doses of gamma radiation.

Table 1. Hematological alterations in peripheral blood of mice with or without A. vasica extract treatment following 8.0Gy

gamma radiation

Post-irradiation time Group Hematological parameters

RBC� 106/mm3 WBC� 103/mm3 Hb gm/100ml Hct %

6h Con 4.8570.27*** 4.070.04*** 9.9670.11*** 30.3070.28***

Exp 6.9670.08*** 5.1870.01*** 13.9070.05*** 34.1270.39***

24 h Con 4.7670.09*** 3.8570.05*** 9.6670.09*** 29.2470.35***

Exp 7.0070.16*** 5.2470.09*** 14.1070.13*** 35.0470.43***

15 day Con 5.2470.02*** 3.9770.05*** 8.8270.11*** 24.3570.33***

Exp 8.4070.07*** 6.0670.03*** 11.3070.10*** 32.7970.66***

30 day Con NS NS NS NS

Exp 9.0070.07 5.7870.62 13.3070.09 43.5670.43

Normal 9.4870.38 6.1870.38 15.3270.22 44.4670.28

AE alone 9.4970.08 6.1870.40 15.2670.80 44.4370.18

Each value represents Mean7SEM. Statistical comparison: Con ¼ 8.0Gy gamma rays: Exp ¼ AE+8.0Gy gamma rays; Normal ¼ No treatment;

AE alone ¼ 800mg/kg/day AE.

Control vs. normal: Experimental vs. control; Significance levels: ***po0.005; NS ¼ Not survived.

A. Kumar et al. / Phytomedicine 12 (2005) 285–293 289

number of RBC gradually increased and reaches tonormal value by day 30 of autopsy interval. WBC countswere observed as significantly higher than control groupof animals. Maximum decline in WBC count wasobserved on 6h autopsy interval (5.1870.10� 103/mm3,po0:005) this value increased and reach to normal valuetill day 15 autopsy interval (6.1670.03� 103/mm3).Hemoglobin level declined to minimum at 6h(13.970.05gm/100ml, po0:005) autopsy interval how-ever, maximum decline was observed at day 15(11.370.10gm/100ml, po0:005). A significant increasewas observed in hematocrit value as compared to controlgroup. However, maximum decline (32.7470.65%,po0:005) was observed on day 15, further increasewas observed and reach to normal on day 30 autopsy(Table 1).

A significant decline was observed in serum alkalinephosphatase activity when exposed 8Gy radiation.Maximum decline was observed at day 15 (2.4670.030KAU po0:005) as compared to normal value (Fig. 5).Serum acid phosphatase value exhibited an increasinglevel. Maximum increase in serum acid phosphataseactivity was observed on 6 h (5.7670.08 KAU,po0:005) autopsy interval and come down to normalvalue (2.9270.05 KAU) till last autopsy interval(Fig. 6).A significant decrease in alkaline phosphatase activity

was observed at 6 and 24 h autopsy interval in AEtreated and irradiated animals. However, increasedvalue of serum alkaline phosphatase was observed onday 15 (5.3470.013 KAU, po0:005) autopsy intervaland reach to normal value till last autopsy interval. The

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Fig. 5. Serum alkaline phosphatase activity in Swiss albino mice after 8.0Gy gamma radiation.

Fig. 6. Serum acid phosphatase activity in Swiss albino mice after 8.0Gy gamma radiation.

Fig. 7. Reduced glutathione (GSH) and LPO levels in Swiss albino mice after 8Gy gamma radiation.

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maximum decline was obtained on 24 h autopsy interval(5.8970.007,po0:005). A significant increase in acidphosphatase activity was observed throughout theexperiment with compared to normal group of animalat each autopsy interval. At 6 h autopsy interval adecline was observed in acid phosphatase activity ascompared to control animals.Blood GSH level did not show significant variation in

AE treated animals. The GSH level observed in AEtreated animal group was (3.58370.31 mg/ml). The

maximum decrease in blood GSH was observed inanimals exposed to 8Gy gamma radiation(2.33670.13 mg/ml). AE treated and irradiated animalsshowed a significant increase in GSH level over thecontrol group of animals but this value remain belownormal (Fig. 7). The significant rise in LPO level wasobserved in control group of animals. The LPO level inAE treated and irradiated group of animals wassignificantly below than the respective control group(Fig. 7).

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Discussion

The radioprotective effect of A. vasica leaf extract wasdemonstrated by determining the LD50/30 (DRF ¼ 1.6).A significant radioprotection was achieved when A.

vasica leaf extract was given orally (800mg/kg bodyweight/day) for 7 consecutive days prior to irradiation.It has been evident that damage to hematopoietic systemis a major factor in the mortality following acuteradiation exposure (Casarett, 1968). In the radiationdose-range studied, hematopoietic as well as gastro-intestinal damage may contribute to mortality. In thepresent investigation, it was observed that 100% ofanimals died when exposed to 8Gy (within 25 days).Such mortality can be attributed to hematopoietic aswell as GI syndrome, radiation damage to gastro-intestinal epithelium cannot be expected in contributingto mortality in mice surviving more than 7 days afterirradiation, since, restoration of the epithelium shouldbe completed by this time (Potten, 1990). One of thecommon features of radiation induced gastro-intestinalsyndrome is marked loss of water and electrolytes whichmay contribute to the weight loss (Griffiths et al.,1999).In the present study, a significant loss in body weight

was evident in control animals (Radiation alone). Theweight loss in the initial phase may be probably due tothe gastro-intestinal damage following irradiation(Smith et al.,1952; Quastler, 1956). The weight lossduring second phase was associated with the decrease inwater intake by animals (Nakamura et al., 1968). Thedose-dependent weight loss in the mouse has also beenreported (Chapman and Jerome, 1956; Shinoda etal.,1967). A. vasica leaf extract pretreated irradiatedanimals showed consistent recovery in body weight andreached to normal by day 30 post-irradiation.In the present study, a significant deficit in hemato-

logical constituents of peripheral blood of controlanimals (Radiation alone) was observed. The decreasein hematological constituents may be attributed to adirect damage by radiation dose (Samarth et al., 2001).Although 3Gy total body dose is required to produce adetectable depression in total red blood cells, wholebody irradiation of moderate dose range (5–10Gy) leadsto a decreased concentration of all the cellular elementsin blood. This can be due to direct destruction of maturecirculating cells, loss of cells from the circulation byhemorrhage or leakage through capillary walls and lossof production of cells (Casarett, 1968). The results of thepresent study indicate that A. vasica leaf extractpretreatment has also provided protection against thehematopoietic death. Thus, enhanced survivability wasobserved in A. vasica leaf extract pretreated mice.It is well known that free radicals generated during

radiolysis of water play the most significant role in theindirect biological damage induced by ionizing radiation(Hall, 1978). GSH offers protection against oxygen

derived free radicals and cellular lethality followingexposure to ionizing radiation (Biaglow et al., 1987).The present study demonstrates a significant reductionin blood GSH, following radiation exposure. This couldbe due to the enhanced utilization of the antioxidantsystem as an attempt to detoxify the free radicalsgenerated by radiation. The depletion of GSH promotesgeneration of reactive oxygen species and oxidativestress with cascade of effects thereby affecting functionalas well as structural integrity of cell and organellemembranes (De Leve et al., 1996). The lower depletionof blood GSH in the A. vasica leaf extract pretreatedirradiated animals could be due to the higher availabilityof GSH, which increases the ability to cope up with thefree radicals produced by radiation.The increased GSH level suggests that protection by

A. vasica leaf extract may be mediated through themodulation of cellular antioxidant levels. GSH is aversatile protector and executes its radioprotectivefunction through free radical scavenging, restorationof the damaged molecule by hydrogen donation,reduction of peroxides and maintenance of proteinthiols in the reduced state (Bump and Brown, 1990). Asthe principal mechanism of the radiation damage is theproduction of free radicals and the scavenging of thesefree radicals by conjugation Phase II enzymes results inradioprotection. Singh et al. (2000) showed that hydro-alcoholic extract of A. vasica modulate the Phases I andII enzyme system and thus result in cancer chemopre-vention in mice. Shimoi et al. (1996) concluded thatplant flavonoids which show antioxidant activity in vitro

also function as antioxidants in vivo, and their radio-protective effect may be attributed to their radicalscavenging activity.The basic effect of radiation on cellular membranes is

believed to be the peroxidation of membrane lipids.LPO can be initiated by radiolytic products, includinghydroxyl and hydroperoxyl radicals (Raleigh, 1987). Itwas observed that, although A. vasica leaf extracttreatment did not significantly alter the LPO level inunirradiated animals but, A. vasica leaf extract pretreat-ment significantly lower the radiation induced LPO interms of malondialdehyde. Inhibition of LPO inbiomembranes can be caused by antioxidants (Koningsand Drijver, 1979; Konings and Osterloo, 1980). It hasbeen shown that more a-tocopherol is needed in themembranes to protect polyunsaturated fatty acids(PUFA) against radiation induced LPO when lowdose-rates are applied (Konings et al., 1979).Although, serum acid phosphatase activity in A.

vasica leaf extract pretreated irradiated animals wasmeasured significantly lower as compared to control(Radiation alone) but found to be higher than thenormal. This suggest that A. vasica leaf extract may helpin causing early recovery by rapid removal of cellulardebris from the tissue, collected due to radiation

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damage. Acid phosphatase is a lysosomal enzyme whichhydrolyses the ester linkage of phosphate ester and helpsin the autolysis of the degenerated cells. On the otherhand, alkaline phosphatase, a brush border enzymesplits various phosphate esters in an alkaline mediumand mediates membrane transport (Godfisher et al.,1973). Thus, increase in these enzymes suggests that acidphosphatase helps in early recovery from radiationdamage by removing debris and alkaline phosphatasehelps in stabilizing the membrane. The results from thepresent investigation indicate that the A. vasica leafextract pretreatment protects against radiation damageby inhibiting radiation induced GSH depletion, decreas-ing LPO level and by increasing phosphatases activityin mice.The results from the present study suggest that

hematopoietic stem cells can be protected from radia-tion induced free radical damage by A. vasica leafextract, which was evident in hematological constituentsin peripheral blood in A. vasica leaf extract pretreatedirradiated mice. Thus the radioprotective effects of AEobserved in the present study can be attributed to theantioxidant properties of A. vasica due to its chemicalconstituents.

Acknowledgments

Authors are thankful Prof. D.P. Agarwal, Head, Dr.K.S. Jheeta and A.A. Chougule (RSO), Department ofRadiotherapy, SMS Medical College and Hospital,Jaipur for providing radiation facility and dosimetry,respectively. The financial support in the form ofResearch Associateship to R.M.S. from CSIR, NewDelhi, India is gratefully acknowledged.

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