Toxicology Letters 132 (2002) 19–25

Syzygium cumini (Jamun) reduces the radiation-inducedDNA damage in the cultured human peripheral blood

lymphocytes: a preliminary study

Ganesh Chandra Jagetia *, Manjeshwar Shrinath BaligaDepartment of Radiobiology, Kasturba Medical College, Manipal 576 119, Karnataka, India

Received 24 October 2001; received in revised form 23 January 2002; accepted 24 January 2002

Abstract

The effects of various concentrations (0.0, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 �g/ml) of the leaf extract ofSyzygium cumini Linn. or Eugenia cumini (SC; black plum, Jamun, family Myrtaceae) was studied on the alterationin the radiation-induced micronuclei formation in the cultured human peripheral blood lymphocytes. Treatment oflymphocytes to various concentrations of SC resulted in a dose dependent increase in the micronuclei-induction,especially after 25–100 �g/ml extract. The exposure of human lymphocytes to various concentrations of SC extractbefore 3 Gy �-irradiation resulted in a significant decline in the micronuclei-induction at all the drug doses whencompared with the non-drug treated irradiated cultures. A nadir in MNBNC frequency was observed for 12.5 �g/mldrug concentration, where the MNBNC frequency was approximately fourfold lower than that of the non-drugtreated irradiated cultures. Therefore, this dose may be considered as an optimum dose for radiation protection. Ourstudy demonstrates that the leaf extract of S. cumini, a plant traditionally used to treat diabetic disorders protectsagainst the radiation-induced DNA damage. © 2002 Published by Elsevier Science Ireland Ltd.

Keywords: Syzygium cumini ; Eugenia cumini ; Lymphocytes; Micronuclei; Gamma radiation; Radioprotection

www.elsevier.com/locate/toxlet

1. Introduction

Since the pioneering work of Patt et al. (1949)that cysteine protected mice and rats against theradiation-induced sickness and mortality, severalchemical compounds and their analogues havebeen screened for their radioprotective ability(Sweeney, 1979). However, the practical appli-

cability of the majority of these synthetic com-pounds remained limited, owing to their hightoxicity at their optimum protective doses. There-fore, it is natural to search for alternatives to thesynthetic compounds. The plant kingdom is vastand the preparations from the plants have beenused to treat several ailments in man since thetime immemorial. In the Indian system ofmedicine, the Ayurveda, a wealth of informationis available about the medicinal properties ofplants and the plant-derived products form anintegral part of the treatment of various disordersin man.

* Corresponding author. Tel.: +91-8252-571201–572000x22814/22122/22815/22816; fax: +91-8252-570062/571927.

E-mail address: gc.jagetia@kmc.manipal.edu (G.C. Jagetia).

0378-4274/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd.

PII: S0378-4274(02)00032-2

G.C. Jagetia, M.S. Baliga / Toxicology Letters 132 (2002) 19–2520

In the last decade and a half the investigatorshave diverted their attention towards plants orplant derived products to look for the agents,which can afford radioprotection at non-toxicdose levels. The plant extracts of Ocimum sanc-tum, Panax ginseng, Chlorella �ulgaris (Jagetia etal., 1986; Zhang et al., 1987; Singh et al., 1995b)have been reported to protect mice against theradiation-induced mortality. Similarly, Spirulinaplatensis, a blue-green alga, garlic, Phylanthusniruri (Qishen et al., 1989; Singh et al., 1995a;UmaDevi et al., 2000) have been reported toprotect mice against the radiation-induced mi-cronucleus formation in the bone marrow. Ex-tracts of O. sanctum and Ginkgo biloba (EGB 761)have also been reported to reduce the radiation-induced chromosomal damage in mice and rats,respectively (Ganasoundari et al., 1997; Alaoui-Youssefi et al., 1999). The herbal preparations likeLiv. 52 and abana have been reported to protectmice bone marrow cells against the radiation-in-duced chromosome damage and micronuclei for-mation (Jagetia and Ganapathi, 1989, 1991;Jagetia and Aruna, 1997). Recently, triphala (amixture of fruits of three plants namely Termina-lia chebula, Terminalia bellerica and Phyllanthusemblica in equal proportions) has been reportedto protect mice against the radiation-inducedmortality (Jagetia et al., 2002).

Syzigium cumini Linn. Skeels is a, medium sizedto large tree, and it has been attributed to possesseveral medicinal properties in the folklore systemof medicine (Warrier et al., 1996). The bark of theplant is astringent, sweet, refrigerant, carminative,diuretic, digestive, antihelminthic, febrifuge, con-stipating, stomachic and antibacterial. The fruitsand seeds are used to treat diabetes, pharyngitis,spleenopathy, urethrorrhea and ringworm infec-tion. The leaves are antibacterial and used tostrengthen the teeth and gums (Warrier et al.,1996). The leaves have been extensively used totreat diabetes, constipation (Bhandary et al.,1995), leucorrhoea, stomachalgia, fever, gastropa-thy, strangury, dermopathy (Warrier et al., 1996)and to inhibit blood discharges in the faeces(Bhandary et al., 1995). Recently, the plant hasbeen reported to poses acetyl oleanolic acid,triterpenoids, ellagic acid, isoquercitin, quercetin,

kaempferol and myricetin (Rastogi and Mehrotra,1990) in different concentrations. Most of thesecompounds have been reported to possess antioxi-dant and free radical scavenging activities (Korinaand Afanaev, 1997; Tanaka, 1994). The diversemedicinal properties attributed to the plant andthe presence of antioxidant and free radical scav-enging compounds in the jamun stimulated us toinvestigate its radioprotective activity. Therefore,the aim of the present study was to evaluate theradioprotective activity of various concentrationsof the leaf extract of Syzygium cumini (SC) in thecultured human peripheral blood lymphocytes ex-posed to 3 Gy of �-radiation.

2. Materials and methods

The leaves of the tree S. cumini Linn. Skeels,(or Eugenia cumini Linn. Druce) family Myrtaceaewas collected locally during the month of April–May of the year, cleaned, shade dried and pow-dered. One hundred grams of the leaf powder wasextracted with dichloromethane and methanol(1:1) as described by Cragg et al. (1997). Anapproximate yield of 11% was obtained. The ex-tract was freeze-dried and stored at −80 °C tillfurther use.

3. Drugs and chemicals

The extract was dissolved in dimethyl sulphox-ide (DMSO) at a concentration of 10 mg/mlimmediately before use and diluted to the re-quired doses. Cytochalasin-B (Sigma Cat. no. C-6762) was dissolved in DMSO at a concentrationof 5 mg/ml, stored at −80 °C and diluted withphosphate buffered saline (PBS) immediately be-fore use. Cytochalasin-B, MEM, L-glutamine,gentamycin sulphate, fetal calf serum (FCS) andDMSO were procured from Sigma Chemical Co.,St. Louis, USA.

3.1. The lymphocyte culture

The whole blood was collected from a healthynon-smoking donor in the heparinised vacutainers

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(Becton Dickinson, USA). The details of thelymphocyte culture are given elsewhere (Jagetia etal., 2001). Briefly, the erythrocytes were allowedto sediment and the buffy coat containing nucle-ated cells was used for the lymphocyte culture.Usually, 106 nucleated cells were inoculated intoeach culture tube containing RPMI-1640 mediumsupplemented with 10% FCS, L-glutamine andphytohemagglutinin (PHA) as the mitogen. Thecultures were divided into two groups as follows.

3.2. SC+sham-irradiation

Immediately after initiation of the lymphocyteculture, the cells were exposed to 0.0, 1.56, 3.125,6.25, 12.5, 25, 50, 100 �g/ml of SC.

3.3. SC+ irradiation

The cultures of this group were treated with 0,1.56, 3.125, 6.25, 12.5, 25, 50/100 �g/ml of SCbefore irradiation.

3.4. Irradiation

Thirty minutes after SC treatment, the cells ofboth groups were exposed to zero (SC+sham-ir-radiation) or 3 Gy (SC+ irradiation) 60Co �-radi-ation from a Tele Cobalt therapy source(Gammatron, Siemens, Germany) at a dose rateof 1.16 Gy/min. Immediately after exposure(within 5 min), the cell cultures were transferredto a CO2 incubator and allowed to grow up to 72h at 37 °C.

3.5. Micronucleus assay

The micronuclei assay was carried out accord-ing to the method of Fenech and Morley (1985)and the protocol described by Krisch-Volders etal. (2000) for in vitro micronucleus assay wasfollowed. Briefly, 5 �g/ml of cytochalasin-B wasadded to the each culture tube 44 h after theirradiation of the cultures to inhibit cytokinesis.The cultures were harvested 72 h after irradiationby centrifugation. The lymphocytes were sub-jected to a mild hypotonic treatment, centrifugedand fixed in Carnoy’s fixative (3:1 methanol, ace-

tic acid). The cells were centrifuged again, resus-pended in a small volume of fixative and spreadon to precleaned coded slides to avoid observer’sbias. Triplicate cultures were used for each drugconcentration for each group.

The slides containing cells were stained with0.125% acridine orange (BDH, England, GurrCat. no. 34001 9704640E) in Sorensen’s buffer(pH 6.8), and washed twice in the buffer. Thebuffer mounted slides were observed under afluorescent microscope equipped with 450–490nm BP filter set with excitation at 453 nm (CarlZeiss Photomicroscope III, Germany) using a40× neofluar objective for the presence of mi-cronuclei (MN) in the binucleate lymphocytes(BNC). A minimum of thousand BNC with well-preserved cytoplasm was scored from each cultureand the frequency of micronucleated binucleatecells (MNBNC) was determined. The micronucleiidentification was done according to the criteria ofCountryman and Heddle (1976), Krisch-Volderset al. (2000).

The statistical significance among various treat-ments between both the groups was determinedusing one-way ANOVA. The Solo 4 statisticalpackage (BMDP Inc., USA and Ireland) was usedfor statistical analysis.

4. Results

The effect of various concentrations of SC onthe radiation-induced micronuclei formation isshown in Table 1. The frequency of MNBNC didnot show significant change up to a dose of 12.5�g/ml when compared to the spontaneous fre-quency of MNBNC. A further increase in thedrug dose resulted in a dose dependent increase inthe frequency of MNBNC up to 100 �g/ml, thelast dose of SC studied.

The treatment of cultures with DMSO (vehicle,zero dose of SC) did not alter the control fre-quency of MNBNC as can be seen from the Table1. Similarly, treatment of lymphocyte with onlyDMSO (0 dose of SC) before exposure to 3 Gyradiation did not alter frequencies of MNBNCsignificantly when compared with the non-DMSOtreated 3 Gy irradiation (data not shown). The

G.C. Jagetia, M.S. Baliga / Toxicology Letters 132 (2002) 19–2522

treatment of lymphocytes with various concentra-tions of SC before exposure to 3 Gy radiationresulted in a significant decline in the frequency ofMNBNC, with respect to non-drug treated irradi-ated cultures. However, the differences among the1.56, 3.12 and 6.25 �g/ml SC was non-significant(Table 1). A drastic decline in the MNBNC induc-tion was observed at a dose of 12.5 �g/ml SC,where this decline was 3.6-fold when compared tothe non-drug treated 3 Gy irradiated cultures.With the further increase in the drug concentra-tion a significant elevation in MNBNC frequencywas observed with respect to 12.5 �g/ml in theSC+ irradiation group that continued up to adose of 100 �g/ml SC eventhough the frequencyof MNBNC was lower (twofold for 25 �g/ml and1.4-fold for 50 and 100 �g/ml, respectively) thanthe 3 Gy irradiation (Table 1). All the dosesprovided significant protection against the radia-tion-induced MNBNC formation. The degree ofprotection against the radiation-induced MNBNCwas highest for 12.5 �g/ml and declined there-after. The frequency of MNBNC was lowest for12.5 �g/ml than the irradiated non-drug treatedcultures and hence this dose of SC has beenconsidered to be the best protective dose.

5. Discussion

Since the discovery of deleterious effects ofradiation, the efforts have been directed to miti-gate the radiation-induced sufferings. Several syn-thetic compounds have been used asradioprotectors and thiol compounds initiallylooked highly promising, however, their practicalapplicability remained limited owing to their hightoxicity at the optimum protective dose (Sweeney,1979). The information on the use of plant ornatural products for their radioprotective capabil-ity is limited. The present study is an attempt inthis direction, where the leaf extract of S. cuminihas been evaluated for its radioprotective activityin the cultured human peripheral bloodlymphocytes.

Treatment of lymphocytes with various dosesof SC before irradiation resulted in a significantdecline in the MNBNC, however, the highestdecline in the MNBNC frequency was observedfor 12.5 �g/ml, where an approximately 3.6-foldreduction in MNBNC was observed when com-pared with the non-drug treated 3 Gy irradiatedcultures. However, with increasing dose of SC theradioprotective action declined (2.5-fold for 25�g/ml and 1.4-fold for 50 and 100 �g/ml, respec-

Table 1Effect of various doses of S. cumini on the radiation-induced micronuclei formation in the cultured human peripheral bloodlymphocytes exposed to 3 Gy of �-radiation

Frequency of MNBNC per 1000�SEMDrug dose (�g/ml)

One MN �Two MN Total MN

SC+SRSC+IRSCSC+IR SC+IRSC+SR

0.33�0.33 66.84�2.7814.20�1.53 14.33�1.810 214.30�4.31147.46�1.62b

1.6 148.72�7.53b21.36�1.84 0.37�0.37 1.27�0.64b,c 23.68�0.28 136.22�3.53b,c

16.53�1.39 130.43�2.96b,c149.28�4.12b 0.37�0.373.125 0.57�0.57b,c16.16�1.7013.40�0.72 142.51�4.13b,c163.21�7.78b 1.11�0.656.25 0.63�0.32b,c12.28�0.75

59.37�2.62b,c13.83�0.670.34�0.34b,c0.33�0.3312.5 57.37�6.84b12.24�2.3133.10�2.17a 86.18�6.34b,c92.80�6.54b 0.56�0.37 1.33�0.88b,c25 33.54�2.4432.44�1.15a 154.62�2.14b 4.81�2.1350 15.00�2.84b,c 38.54�3.94 148.40�3.93b,c

27.49�1.76b,c18.70�2.90a142.52�3.97b60.29�5.60a100 149.18�4.59b,c94.77�3.89

SC, Syzigium cumini extract; SR, sham-irradiation; IR, irradiation. Absence of symbols, non-significant.a P�0.05 when spontaneous MNBNC frequencies are compared with various doses of SC in SC+sham-irradiation.b P �0.001 the sham-irradaiton is compared SC+irrradiation.c P�0.0001 the first value (3 Gy irradiation without SC treatment) of the column No. 5 and 6 compared with the remaining values

(1.56, 3.125, 6.25, 12.5, 25, 50 and 100 �g/ml SC) of the same column.

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tively). The earlier studies on radioprotectionhave shown that an agent in test (for radioprotec-tive action) acts only at a particular dose andabove, which it may not be protective and caneven be toxic (Thomson, 1962). The reason maybe that after a particular concentration, a com-pound may start manifesting its toxic effects. Theactive principle of Plumbago rosea, theplumbagin, has been reported to stimulate thegranulocytes in vitro at pico to femto gram range,while at higher doses it results in the immunosup-pressive activity (Wagner et al., 1988). A similaraction cannot be ruled out for SC that has offeredan optimum protection at 12.5 �g/ml SC extract,where the MNBNC frequency was the lowestcompared to other doses of the extract in theSC+ irradiation group while, the higher dosesresulted in the decline in the protective action ofSC. As far as authors are aware, there are noreports regarding the use of SC extract as theradioprotective agents in vitro and this is the firstreport of use of SC for its radioprotective activity.The other natural products like vitamin A, C andE that are present in several vegetables has beenreported to reduce the radiation-induced micronu-clei formation in the cultured human peripheralblood lymphocytes exposed to gamma rays(Gasiev et al., 1996). �-Carotene has also beenreported to prevent the X-ray induced micronucleiinduction (Umegaki et al., 1994).

The extracts of S. platensis, a blue-green alga,garlic and P. niruri have been reported to protectmice bone marrow against the radiation-inducedmicronucleus formation (Qishen et al., 1989;Singh et al., 1995b; UmaDevi et al., 2000) Ex-tracts of the plants O. sanctum and G. biloba(EGB 761) have also been reported to reduce theradiation-induced chromosomal damage in miceand rats, respectively (Ganasoundari et al., 1997;Alaoui-Youssefi et al., 1999). Similarly, the plantformulations like Liv. 52 and abana have alsobeen reported to protect mice bone marrow cellsagainst the radiation-induced chromosome aber-rations and micronuclei formation (Jagetia andGanapathi, 1989, 1991; Jagetia and Aruna, 1997).

Certain plant flavonoids like luteolin, quercetin,kaempferol, morin, rutin, fisetin, myricetin, epi-catechin, genistein, eridictyol, phloretin have been

found to protect mice bone marrow cells againstthe radiation-induced micronuclei (Shimoi et al.,1994, 1996, 1997). The flavonoids of O. sanctum,orientin and vicenin have also been reported toprotect mice bone marrow cells against the radia-tion-induced chromosome damage and mortality(Uma Devi et al., 1998, 1999).

The BNC with �2 MN were significantly re-duced in the lymphocytes treated with SC beforeexposure to 3 Gy of �-radiation, indicating theinhibition of multiple damage to DNA and com-plex chromosome aberrations by SC. This indi-cates the efficacy of SC extract in reducing theDNA damage and complex chromosomal aberra-tions. The complete absence of BNC with �2MN for 12.5 �g/ml SC indicates that the presenceof SC before irradiation has completely inhibitedmultiple sites of DNA damage and complex chro-mosome aberrations. A similar reduction in PCEbearing 2 MN has been observed in mice bonemarrow treated with (E) 4-[4-N,N-dimethy-laminophenyl]but-3-en-2-one, a synthetic com-pound (Jagetia et al., 1994) and the extract ofabana (Jagetia and Aruna, 1997).

Myricetin, has been reported to increase theexpression of DNA polymerase beta gene in adose dependent manner, an enzyme responsiblefor the error-free DNA repair (Abalea et al.,1999). Since SC contains myricetin the reductionin the radiation-induced micronuclei by SC maybe owing to the error-free DNA repair in theSC+ irradiation group.

The exact mechanism of action of SC extract isnot known. However, it is plausible that scaveng-ing of free radicals by SC extract may have playedan important role in providing the protectionagainst the radiation-induced damage to theDNA. This contention is supported by the obser-vation that flavonoids like quercetin, kaempferoland myricetin, which are present in the SC (Ras-togi and Mehrotra, 1990) have been reported toscavenge free radicals like OH and superoxide andalso inhibits the lipid peroxidation (Maridonneau-parini et al., 1986; Uddin and Ahmad, 1995;Korina and Afana’ev, 1997; Abalea et al., 1999).Kaemperol and quercetin have been reported tosuppress the cytotoxicity of superoxide ion andhydrogen peroxide in Chinese hamster V79 cells

G.C. Jagetia, M.S. Baliga / Toxicology Letters 132 (2002) 19–2524

(Nakayama et al., 1993; Nakayama, 1994). Thepolyphenol ellagic acid, which is also present in S.cumini has been reported to be antimutagenic,chemopreventive (Tanaka, 1994), antioxidant andit has also been found to inhibit the radiation-in-duced lipid peroxidation in the liver of mice(Theresiamma et al., 1996). The presence offlavanoids and ellagic acid in SC extract mighthave been responsible for the observed radiopro-tection in the lymphocytes. The further studies arebeing undertaken to investigate the active princi-ple(s) and their mode of action.

Acknowledgements

We thank Professor M.S. Vidyasagar, and Dr J.Velumurugan, Department of Radiotherapy andOncology, Kasturba Medical College, Manipal,India for providing the necessary irradiation facil-ities and help in radiation dosimetry, respectively.

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