Food Chemistry 126 (2011) 39–45

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Food Chemistry

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Antioxidant and radioprotective properties of commercially grown litchi(Litchi chinensis) from India q

Sudhanshu Saxena, Sachin N. Hajare, Varsha More, Sanjeev Kumar, Surbhi Wadhawan, B.B. Mishra,Madan Narayan Parte, Satyendra Gautam, Arun Sharma ⇑Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400 085, Maharashtra, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2010Received in revised form 20 August 2010Accepted 12 October 2010

Keywords:Litchi chinensisAntioxidantRadioprotectionCell survivalPhenolics

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.051

q All authors have equally contributed.⇑ Corresponding author. Tel.: +91 22 2559574

25505151/25505150.E-mail address: ksarun@barc.gov.in (A. Sharma).

In an earlier study, radiation treatment (0.5 kGy) and low temperature (4 �C) storage were found toextend the shelf life of litchi fruit var. ‘Shahi’ and ‘China’ from India up to 28 days, while maintainingits physical, biochemical and organoleptic properties. The current study was aimed to elucidate the anti-oxidant and radioprotective properties of the litchi fruit, and the effect of radiation processing on theseproperties. The litchi fruit was found to be significantly rich in antioxidant and radioprotective properties,and the antioxidant parameters were found to be well correlated with the phenolic and flavonoid con-tents. A significant protection to pBR322 plasmid DNA and Escherichia coli cells from radiation induceddamage was observed in the presence of litchi juice. Plasmid DNA was well protected even at the doseof 5 kGy, whereas, bacterial cells could be protected up to 0.5 kGy. Neither of these properties was foundto be sensitive to radiation processing or low temperature storage.

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1. Introduction

Litchi (Litchi chinensis) is a tropical, non-climacteric fruit,belonging to the Sapindaceae family and is known to have origi-nated from South-East Asia (Rivera-López, Ordorica-Falomir, &Wesche-Ebeling, 1999). The fruit has bright red and attractive peri-carp surrounding a white and translucent fleshy aril, having sweetand pleasant flavour. The juicy fruit can be eaten directly and canalso be used to manufacture juice, vinegar, jelly and wine, or usedin ice creams and sorberts (Salunkhe & Desai, 1984).

In general, fruits and vegetables are known to contain a widevariety of phytochemicals, such as polyphenols, carotenoids andvitamin C. More than 5000 individual phytochemicals have beenidentified in fruits, vegetables, and grains (Liu, 2003). Some ofthese contribute to the antioxidant potential of fruits and vegeta-bles. Ascorbic acid and phenolics are known as hydrophilic antiox-idants, whereas carotenoids are known as lipophilic antioxidants(Halliwell, 1996). Oxidative stress caused by free radicals is in-volved in the aetiology of a wide range of chronic diseases, suchas cancer, cataract, cerebral pathologies, rheumatoid arthritis, agerelated macular degeneration and other degenerative diseases.This happens because free radicals are chemically highly reactive

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and could cause oxidative damage to important cellular macromol-ecules, such as nucleic acids, proteins and lipids (Clifford, 1995).Epidemiological studies have indicated that frequent consumptionof natural antioxidants could lower the risk of chronic diseases(Temple, 2000). Thus, the natural antioxidants present in fruitsand vegetables could scavenge free radicals and provide radiopro-tective ability to these foods. In case of litchi, previous studies weremainly focused on antioxidant activities of the pericarp and seed,which were found to have high level of phenolics, including gallicacid, flavonoids, such as epicatechin and epicatechin gallate, andpigments, such as anthocyanidins and procyanidins, contributingto its high antioxidant property (Lee & Wicker, 1991; Prasad, Yang,Ruenroengklin, Zhao, & Jiang, 2009; Prasad, Yang, Yang, et al.,2009). However, surprisingly, there is very little information avail-able on the antioxidant activities of edible part (aril) of litchi,which bears obviously more significance.

India is the second largest producer of litchi after China, involv-ing an area of 62,000 Ha and a total annual production of 0.5 mil-lion tones (Hajare et al., 2010). India accounts for about one-fifthof the global production and has a good export potential. In India,Bihar is the leading state for litchi production, which accounts forthree quarters of the total production of the country. The agro-cli-matic conditions of the state are ideal for cultivation of this fruit.Assam, Orissa, Punjab, Tripura, Uttarakhand and West Bengal arethe other main litchi growing states.

Although India is the second largest producer of litchi, verylimited information is available on the antioxidant properties of

40 S. Saxena et al. / Food Chemistry 126 (2011) 39–45

Indian litchi cultivars. Among the different litchi cultivars that aregrown in India, ‘Shahi’ and ‘China’ are the two major commerciallygrown cultivars (Hajare et al., 2010). ‘Shahi’ is a popular and anearly maturing variety. The fruits of ‘Shahi’ are round deep red col-oured and have aromatic pulp, whereas, the variety ‘China’ is latematuring, and the plants are comparatively dwarf. The fruits of‘China’ are large, conical, deep red in colour and contain high ratioof pulp.

In a recent study, Indian litchi (var ‘Shahi’ and ‘China’) weretreated using low dose gamma radiation (0.5 kGy) and were storedat 4 �C, which helped in extending its shelf life up to 28 days, byreducing the microbial load and inhibiting the post-harvest physi-ological spoilage (Hajare et al., 2010). Radiation processing of foodis a physical process in which food and agricultural commoditiesare exposed to radiation to achieve desirable effects, such as theinhibition of sprouting, delay in ripening and the killing of insectpests, parasites, and pathogenic and spoilage microorganisms(Heather & Hallman, 2008). Ionising radiation treatment has beenknown to extend the post-harvest life of many tropical and sub-tropical fruits by virtue of inhibiting insect infestation and physio-logical changes (Singh & Pal, 2009). In many countries, theprocessing by ionising radiation is an approved phytosanitarytreatment against most of the insect pests associated with freshhorticultural produce (Hajare et al., 2010; Heather & Hallman,2008; Singh & Pal, 2009).

Radiation processing of Indian litchi fruits too helped in over-coming the quarantine barrier which would be helpful in facilitat-ing its export. Therefore, the major objective of the currentresearch was to evaluate the antioxidant and radioprotective prop-erties of fruits of the Indian litchi cultivars ‘Shahi’ and ‘China’.Moreover, the effect of gamma radiation processing on these qual-ity attributes was also examined.

2. Materials and methods

2.1. Procurement of fruits and radiation processing

Fruits of Indian litchi cultivars ‘Shahi’ and ‘China’ were procuredfrom a local market within 6–8 h of harvest. A chain of govern-ment-recognised wholesale and retail suppliers were involved inthe process of supplying the fruits from the field to the market,which assured the overall authenticity of the samples. Preliminaryscreening was done to eliminate damaged or diseased fruits. Later,the fruits were packaged (10 fruits per packet) and sealed in low-density polyethylene bags (LDPE, thickness: 700 gauge; water va-pour transmission rate: 0.4 g/m2/day; oxygen transmission rate:1800 cm3/m2/day). The packaged samples were irradiated at0.5 kGy dose of gamma radiation and stored at 4 �C. Gamma irradi-ation was performed at ambient temperature (26 ± 2 �C) in a co-balt-60 based Food Package Irradiator (AECL, Canada; activity1.97 PBq; dose rate 2.4 kGy/h) at Food Technology Division, Bhab-ha Atomic Research Centre, Mumbai, India. Dosimetry was per-formed using ceric-cerous sulphate dosimeters (ASTM, 1993).

2.2. Extraction of litchi juice for biochemical analysis

For analysis, the edible portion (aril) from total 30 fruits (10fruits each in three replicates) of both varieties were ground indi-vidually in a mixer grinder, after manually removing the peel andthe seed from the fruit. The homogenate was subsequently filteredthrough two layered muslin cloth and spun at 10,000g for 10 minin a centrifuge (Tigra, model: CMF 15 KR, Poland) at ambient tem-perature, to remove any suspended particulate matter that couldaffect the antioxidant and radioprotective assays by interferingwith the spectrophotometric measurements, as well as the DNA

gel electrophoresis, respectively (Patrineli, Clifford, Walker, & Ioan-nides, 1996; Rodriguez-Saona, Fry, McLaughlin, & Calvey, 2001).The supernatant obtained was designated as clear juice, whichwas used during different biochemical analyses. For both varieties,all the analyses were performed in three independent sets, each sethaving three replicates.

2.3. Assays of antioxidant activities

2.3.1. DPPH radical scavenging activityThe 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

effect (H/e� transferring ability) of litchi juice was measured asper the method described by Chen, Lin, and Hsieh (2007). TheDPPH was dissolved in absolute ethanol to a 0.2 mM concentration.This solution is violet in colour that turns to yellow as the molarabsorptivity of the DPPH radical at 517 nm reduces from 9660 to1640 M�1cm�1 when the odd electron of DPPH radical becomespaired with hydrogen from a free radical scavenging antioxidantto form the reduced DPPH-H (Häussler & Becker, 2008). A 50 ll ali-quot of clear litchi juice was diluted to 550 ll with 70% ethanol,and vigorously mixed with 400 ll of DPPH solution by vortexing.The mixture was incubated at ambient temperature for 15 minand the absorbance of the solution (T1) was measured at 517 nmusing a spectrophotometer. The sample blank (B1) consisted of600 ll of 70% ethanol and 400 ll of DPPH, whereas the DPPH blank(B2) contained 50 ll of litchi juice, 550 ll of 70% ethanol and 400 llof absolute ethanol. The DPPH scavenging activity was calculatedusing the following formula:

DPPH scavenging activity ð%Þ ¼ ½1� fðT1 � B2Þ=B1g� � 100

where T1, B1, and B2 are the absorbencies of the sample, sampleblank and DPPH blank, respectively. Moreover, a calibration curveshowing the percentage DPPH scavenging activity using as the ref-erence standard butylated hydroxytoluene (BHT: 0.2–3.7 mM inethanol) was also plotted.

2.3.2. Ferric reducing antioxidant power (FRAP) assayThe reducing power of litchi juice was determined using the

method of Oyaizu (1986). In the reducing power assay, the pres-ence of reductants (antioxidants) in the samples reduce Fe3+ toFe2+ by donating an electron. The amount of Fe2+ complex is mon-itored by measuring the formation of Perl’s Prussian blue at700 nm. A higher absorbance at 700 nm indicates a higher reduc-ing power. For determining the reducing power of litchi juice,1 ml ethanolic extract [1%, 5% and 10% (v/v) in ethanol] was mixedwith 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potas-sium ferri-cyanide (1%). The mixture was incubated at 50 �C for20 min. Then, 2.5 ml of 10% trichloroacetic acid was added, andthe solution was mixed by vortexing. The mixture was centrifugedat 3000g for 10 min. A 2.5 ml aliquot of the supernatant was mixedwith equal amount of milli Q water and 0.5 ml of 0.1% FeCl3. Theabsorbance was measured at 700 nm using a spectrophotometer.In addition, a calibration curve describing the ferric reducing anti-oxidant power using as the reference standard BHT (0.48–7.8 mMin ethanol) was also plotted.

2.4. Escherichia coli survival study upon gamma radiation treatmentin the presence of litchi juice

E. coli strain MG1655 was grown overnight in Luria–Bertanimedium and was diluted to �105 cells/ml using saline (0.85%). Inthe control set, 500 ll of diluted cell suspension was mixed with500 ll of sterile saline (0.85%), whereas in the test, 500 ll of filtersterilized (using 0.45 lm PVDF filter; Millex-HV, Millipore Corp.,Ireland) litchi juice was mixed with the diluted cell suspension.Both the control and test samples were exposed to different doses

S. Saxena et al. / Food Chemistry 126 (2011) 39–45 41

of gamma radiation (0, 0.1, 0.25 and 0.5 kGy). Then, the sampleswere spun at 20,000g for 15 min at 4 �C in a centrifuge, the super-natant was discarded, and the pellet was washed with sterile sal-ine. The cell pellet was resuspended in 1 ml saline by vigorousvortexing. For the enumeration of viable cell counts, the cell sus-pensions were serially diluted using sterile saline (0.85%) and thedilutions were spread plated on Luria–Bertani agar plates. Theplates were incubated at 37 �C for 24 h in an incubator (Sanyo,model: MIR-262, Japan) and the colony forming units (cfu) werecounted. The decimal reduction dose (D10) values were calculatedfrom a linear regression survival curve.

2.5. Evaluation of radioprotective effect of litchi juice

The radioprotective potential of litchi juice was evaluated usingthe method described earlier by Sharma, Gautam, and Jadhav(2000). A 15 ll aliquot of clear litchi juice was mixed with 15 llof ultra purified (using caesium chloride density gradient)pBR322 plasmid DNA (�400 ng) (Bangalore Genei, India) in a ster-ile microfuge tube (Eppendorf, 1.5 ml). The samples were radiationtreated at different doses (1–5 kGy), and subjected to agarose gel(1%) electrophoresis at 4 V/cm, using Tris–acetate–EDTA (TAE) buf-fer (pH 8.0). Then the gel was stained with ethidium bromide(0.5 lg/ml) and visualised using a UV-transilluminator.

2.6. Total phenolic content

The total phenolic content of fruit juice was determined by theFolin–Ciocalteu method (Singleton, Orthofer, & Lamuela-Raventos,1999). A 100 ll aliquot of clear litchi juice was mixed with 700 llof milli Q water and 50 ll of 0.2 N Folin–Ciocalteu reagent. Thesolution was thoroughly mixed by vortexing and further incubatedfor 2 min at ambient temperature. A 150 ll aliquot of sodium car-bonate solution (0.2 g/ml) was added to the reaction mixture andfurther incubated for 2 h at the same temperature. The absorbancewas measured at 765 nm using a spectrophotometer. The totalphenolic content was determined using a standard curve preparedusing gallic acid (0–200 mg/l) and the mean value was expressedas mg of gallic acid equivalents (mg GAE)/g of litchi fruit.

2.7. Statistical analysis

Statistical analysis was performed using BioStat 2009 VersionProfessional 5.8.0.0 (AnalystSoft Inc., Canada). All the biochemicalanalyses were repeated in three sets independently and from eachset, the analysis was performed in triplicate. Means and standarddeviations were calculated taking all the readings. As three fixedfactors (cultivar, radiation dose and storage days) were studied,three-way ANOVA (analysis of variance) was performed. Themeans separation test used was the Fisher LSD. The level of signif-icance at which the analyses were performed was P 6 0.05.

3. Results and discussion

3.1. Antioxidant activities

As in our previous study (Hajare et al., 2010), gamma radiationprocessing at 0.5 kGy and storage at 4 �C was helpful in achievingshelf life extension; the effects of radiation processing and lowtemperature storage on the antioxidant profile of litchi fruits werealso assessed. Fruit juices from ‘Shahi’ and ‘China’ cultivars wereanalysed for their antioxidant content by measuring antioxidantparameters, such as the DPPH radical scavenging activity and theferric reducing antioxidant power. The antioxidant property oflitchi juice was compared with butylated hydroxytoluene (BHT)

as the reference standard. Furthermore, a study was performedto measure the level of probable factor(s) contributing to the over-all antioxidant activity. The end of storage life of the fruits wasdetermined based on physical observations. When visible fungalgrowth and rotting were noticed, the fruits were not consideredsuitable for consumption, and designated as spoiled. During stor-age, for both litchi varieties, the nonirradiated control samplesdid not last more than 15 days at 4 �C (Hajare et al., 2010).

3.1.1. DPPH scavenging activityFig. 1A and B are depicting the DPPH radical scavenging activity

(%) in both ‘Shahi’ and ‘China’ litchi varieties. In fresh, nonirradi-ated ‘Shahi’ fruit juice (effective dilution in the reaction mixture,1:20), the DPPH radical scavenging activity was found to be 89%.This was found to be equivalent to�2.5 mM of BHT. During storagefor 10 days, a slight but insignificant increase was observed for thisantioxidant parameter. In the case of ‘Shahi’ litchi fruits exposedwith a gamma radiation dose of 0.5 kGy, the radical scavengingactivity on day 0 was found to be 92%. Both the nonirradiated aswell as the radiation treated samples were found to have almostsimilar DPPH scavenging activities on day 0 and day 10. No statis-tically significant change was noted until 20 days of storage. How-ever, on the 28th day, the value significantly decreased to 74%(P 6 0.05).

In nonirradiated ‘China’ fruit juice (effective dilution in the reac-tion mixture, 1:20) the DPPH radical scavenging activity was foundto be 85% on day 0 which remained almost constant until 10 daysof storage. This was found to be equivalent to �2 mM of BHT. Forthe ‘China’ variety, the fruits treated with 0.5 kGy dose of gammaradiation, the activities were found to be 85% on day 0. An almostconstant radical scavenging activity was observed until 20 days ofstorage. A significant decrease to 73% was observed in the 28 daysstored sample. Concomitant with ‘Shahi’, the radiation treatmentwas not found to alter the antioxidant property in ‘China’ fruitsas well. The difference in the DPPH radical scavenging activity(%) shown by both these varieties ‘Shahi’ and ‘China’ was foundto be statistically significant (P 6 0.05). Interestingly the ‘Shahi’fruits were found to have comparatively high DPPH scavengingactivity than ‘China’, whereas the vitamin C content was signifi-cantly lesser than ‘China’. The findings indicate that the vitaminC level in a fruit is not only the factor responsible for providingantioxidant potential but other phytochemicals also contribute sig-nificantly. In an earlier report, the antioxidant capacity measuredwith DPPH was generally stable during storage for most of the Bel-gian fruits and vegetables (Kevers et al., 2007). Shivashankara,Isobe, Al-Haq, Takenaka, and Shina (2004) also reported that theantioxidant capacity of mango fruits was maintained during lowtemperature (5 �C) storage for 20 days.

3.1.2. Ferric reducing antioxidant power (FRAP) assayIn this study, both litchi varieties showed considerable antiox-

idant activity in terms of reducing power as shown in Fig. 2A andB. There was a concentration dependent increase in the reducingpower of the litchi samples. A 10% litchi extract exhibited moreabsorbance at 700 nm as compared to 5% and 1% extracts (datanot shown). The reducing power in terms of absorbance at700 nm for fresh nonirradiated ‘Shahi’ fruit juice (effective dilu-tion in the reaction mixture, 1:19) on day 0 was found to be0.22, which was found to be equivalent to �1 mM of BHT. Thereducing power increased to 0.29 on day 10 of storage, whichwas found to be statistically significant (P 6 0.05). In ‘Shahi’ fruitstreated with a gamma radiation dose of 0.5 kGy, the absorbanceon day 0 was found to be 0.28, which showed a statistically sig-nificant increase until 20 days of storage followed by a declineon the 28th day. On day 0 of analysis, the 0.5 kGy treated ‘Shahi’

Fig. 1. DPPH scavenging activity. Mean values were analysed by three-way analysis of variance (ANOVA) and Fisher LSD. a,bDifferent letters on bar indicate significantdifferences among means (P 6 0.05). #Effective dilution of litchi juice in reaction mixture = 1:20.

Fig. 2. Ferric reducing antioxidant power. Mean values were analysed by three-way analysis of variance (ANOVA) and Fisher LSD. a,b,cDifferent letters on bar indicatesignificant differences among means (P 6 0.05). #Effective dilution of litchi juice in reaction mixture = 1:19.

42 S. Saxena et al. / Food Chemistry 126 (2011) 39–45

fruits exhibited a statistically significant increase in absorbance ascompared to nonirradiated fruits (P 6 0.05).

In case of the ‘China’ variety, the fresh nonirradiated fruit juice(effective dilution in the reaction mixture, 1:19) showed an absor-bance value of 0.39 on day 0 of analysis, which was found to beequivalent to �2 mM of BHT. The absorbance value decreasedslightly until 10 days of storage but this decline was found to bestatistically insignificant. For the ‘China’ variety, the fruits treatedat 0.5 kGy dose, the absorbance values were found to be compara-tively lower than the nonirradiated samples on day 0. Moreover, inthe 0.5 kGy treated fruits, the absorbance values remained almostconstant during storage until 28 days. The difference in the FRAPvalues for both the litchi varieties was found to be statistically sig-nificant (P 6 0.05).

3.2. Biochemicals contributing towards antioxidant activity

Different biochemicals, such as phenolics, flavonoid, carotenoid,vitamin C are generally reported to contribute to the antioxidantactivities of different fruits. Two of these, the flavonoid content

and vitamin C, have been analysed in our earlier study and arebeing discussed here to have a comparable correlation analysiswith the antioxidant activities (Hajare et al., 2010). The phenoliccontent of analysed litchi cultivars is shown in Table 1. The pheno-lic content in nonirradiated fresh ‘Shahi’ fruits on day 0 was foundto be 312 lg GAE/g. A slight but insignificant increase to322 lg GAE/g was observed for these fruits on day 10. The phenoliccontent in fruits treated with 0.5 kGy dose of gamma radiation wasfound to be 312 lg GAE/g on day 0. The phenolic content remainedalmost stable in fruits analysed on the 10th day. However, a statis-tically significant increase to 323 lg GAE/g was noted on the 20thday (P 6 0.05). Furthermore, a slight but statistically insignificantincrease to 328 lg GAE/g was observed for the fruits on the 28thday.

The phenolic content of nonirradiated ‘China’ fruits on day 0was found to be 318 lg GAE/g; it remained constant on the 10thday of analysis. The fruits of ‘China’ treated at 0.5 kGy dose of gam-ma radiation showed a slightly higher phenolic level (331 lg GAE/g) as compared to nonirradiated fruits on day 0. But this increasewas found to be statistically insignificant. The phenolic content

Table 1Total phenol content (lg GAEA/g as mean ± SDB) in litchi during storage: ‘Shahi’ and ‘China’.

Radiation dose (kGy) ‘Shahi’ ‘China’

0 day 10 day 20 day 28 day 0 day 10 day 20 day 28 day

0 312a ± 7 322a ± 18 Unusable Unusable 318a ± 7 317a ± 16 Unusable Unusable0.5 312a ± 7 314a ± 7 323a ± 3 328a ± 16 331a ± 12 358a ± 51 311a ± 9 300a ± 8

Mean values were analysed by three-way analysis of variance (ANOVA) and Fisher LSD.a Same letter in superscript indicates statistically insignificant differences among means.

A Gallic acid equivalent.B Standard deviation.

Fig. 3. Protection of E. coli cells by litchi juice against gamma radiation inducedkilling. Mean values were analysed by three-way analysis of variance (ANOVA) andFisher LSD. No cell survival was observed in 0.5 kGy treated cells in the absence ofjuice. a,b,c,dDifferent letters on bar indicate significant differences among means(P 6 0.05).

S. Saxena et al. / Food Chemistry 126 (2011) 39–45 43

was found to increase on the 10th day, but it showed a decreasedtrend both on the 20th as well as 28th day. However, these changeswere found to be statistically insignificant. The difference in thephenolic content for both litchi varieties was found to be statisti-cally insignificant. Moreover, the phenolic content observed in In-dian litchi was found to be slightly higher than Mauritian litchi(Luximon-Ramma, Bahorun, & Crozier, 2003). The total phenoliccompounds remained almost stable during storage for most ofthe Belgian fruits (Kevers et al., 2007). In an earlier report the phe-nolic level did not change in Hayward kiwifruit when stored for2 months at 0 �C (Tavarini, Degl’Innocenti, Remorini, Massai, &Guidi, 2008).

Among the polyphenols, flavonoids are the most common andwidely distributed group of plant phenolic compounds. They arecharacterised by a benzo-y-pyrone structure which is ubiquitousin fruits and vegetables. The flavonoid content in ‘Shahi’ and ‘Chi-na’ was found to be 31.0–31.2 and 21.5–31.0 lg CE/g, respectively(Hajare et al., 2010). Generally, it is known that fruits with highantioxidant capacity have high phenolic content, especially flavo-noids (Connor, Luby, Hancock, Berkheimer, & Hanson, 2002).Rice-Evans, Miller, and Paganga (1996) have very clearly indicatedthat not all plant products bear the same phenolic composition andnot all phenolics possess the same antioxidant capacity. It is notthe quantity but also the quality of polyphenols, which serves asthe major determinant of the antioxidant capacity of food. Robards,Prenzler, Tucker, Swatsitang, and Glover (1999) also reported thatthe antioxidant activity is affected not only by the polyphenoliccontent but also by the compositional change. Therefore, sampleswith similar concentrations of total phenolics may vary remark-ably in their antioxidant activity as can be seen from the differentantioxidant values observed for both litchi varieties.

The level of vitamin C was significantly higher in ‘China’(25 mg%) compared to ‘Shahi’ (17 mg%) (Hajare et al., 2010). Ascor-bic acid is one of the most powerful natural antioxidants and uponinteraction with reactive oxygen species (ROS) it gets oxidised todehydroascorbate via the intermediate ascorbyl free radical (Hen-son, Block, & Levine, 1991).

3.3. Radiation protection of E. coli cells by litchi juice

Fig. 3 demonstrates the protection of E. coli MG1655 cells bylitchi juice from gamma radiation induced damage. There was adose dependent decrease in the cell count observed in E. coli cells.At a gamma radiation dose of 0.5 kGy, there was complete killing ofE. coli cells suspended in saline. However, significantly increasedsurvival was noticed in the cells treated with gamma radiation inthe presence of litchi juice. Even at a dose of 0.5 kGy, 3 log cyclecell survival was observed in the presence of litchi juice for bothvarieties. The major free radicals resulting from aqueous radiolysisare OH, H, e�aq, HO2, H3O+, etc. These free radicals are highly reactiveand are known to react with cellular macromolecules, such as DNA,RNA, proteins and membranes, and cause cell dysfunction andmortality (Dragaric & Dragaric, 1971; Pradhan, Nair, & Sreenivasan,

1973). The presence of litchi juice in cellular milieu is protectingthe cells against radiation mediated damage. The decimal reduc-tion dose (D10) value for E. coli in 0.85% saline was found to be0.1 kGy, and in presence of litchi juice, the value increased to0.27 kGy. Insignificant difference was observed in the level of pro-tection offered by the juice of both litchi varieties. Thus, the find-ings indicated that the phytochemicals present in litchi juicehave strong antioxidant properties capable of significantly scav-enging free radicals generated during gamma radiation treatment,thus leading to increased cell survival.

3.4. Radiation protection of plasmid DNA by litchi juice

Fig. 4A and B present the DNA agarose gel profiles illustratingthe protection of pBR322 plasmid DNA from gamma radiation in-duced damage by litchi juice. Nonirradiated pBR322 plasmid DNAis highly supercoiled (about 80%). When exposed to a 1 kGy doseof gamma radiation there was almost complete degradation ofpBR322 plasmid DNA (Fig. 4A, lane 2), whereas in the presenceof litchi juice, the degradation was significantly inhibited(Fig. 4A, lanes 4–12), thus indicating that litchi juice has strongradioprotective activity. Interestingly, there was presence ofsupercoiled DNA (Fig. 4B, lanes 2, 3, 5, 6, 8, 9) in DNA treatedin the presence of juice from ‘China’ variety, whereas this formof DNA was absent in DNA treated in the presence of ‘Shahi’(Fig. 4, lanes 4:12). This differential behaviour of juice from twodifferent varieties is unclear but it could be attributed to the high-er content of ascorbic acid present in the ‘China’ variety as com-pared to ‘Shahi’ (Hajare et al., 2010). Dani et al. (2007) reportedthat ascorbic acid monoglucoside protected plasmid pBR322DNA from gamma radiation induced strand breaks. Radioprotec-tion by aqueous extracts of chilli, black pepper and turmeric toplasmid DNA has been reported earlier (Sharma et al., 2000).Radiation treatment can as such produce a variety of lesions in

A. ‘Shahi’

B. ‘China’

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10

Fig. 4. Agarose gel electrophoresis displaying radioprotection of pBR322 plasmidDNA by litchi juice. (A) Lane 1: nonirradiated DNA; Lane 2-3: 1 and 2 kGy gammairradiated DNA, respectively; Lanes 4-6: Protection of DNA by juice from nonirra-diated fresh fruits upon gamma radiation treatment [4: 1 kGy; 5: 2 kGy; 6: 5 kGy];Lanes 7: 9 Protection of DNA by juice from fresh fruits treated with 0.5 kGyradiation [7: 1 kGy; 8: 2 kGy; 9: 5 kGy]; Lanes 10-12 protection of DNA by juicefrom 0.5 kGy radiation treated fruits stored for 28 days [10: 1 kGy; 11: 2 kGy; 12:5 kGy]. (B) Lane 1: DNA molecular weight marker; Lanes 2-4 Protection of DNA byjuice from nonirradiated fresh fruits [2: 1 kGy; 3: 2 kGy; 4: 5 kGy]; Lanes 5-7protection of DNA by juice from fresh fruits treated with 0.5 kGy radiation [5:1 kGy; 6: 2 kGy; 7: 5 kGy]; Lanes 8-10 Protection of DNA by juice from 0.5 kGytreated fruits stored for 28 days [8: 1 kGy; 9: 2 kGy; 10: 5 kGy].

44 S. Saxena et al. / Food Chemistry 126 (2011) 39–45

DNA resulting in both single and double stranded breaks, alter-ation of bases, destruction of sugar moiety and cross-linking,and formation of dimers (Kada, Kaneko, Matsuzaki, & Hara,1985). The formation of open circular form of plasmid DNA fromsupercoiled plasmid DNA is an indication of single strandedbreaks (SSBs), whereas the formation of linear form is indicativeof double stranded breaks (DSBs) (Zhang & Omaye, 2001).

3.5. Correlation among the biochemical parameters and theantioxidant assays

A strong positive relationship between the DPPH scavengingactivity and the total phenolic content was observed for both litchivarieties (Tables S1A and S1B). The corresponding correlation val-ues for ‘Shahi’ and ‘China’ were 0.95 and 0.80, respectively. Highercorrelation values were observed between the antioxidant activi-ties and the flavonoid content. Strong correlation coefficients(0.90 and 0.93) were also observed between the FRAP value andthe total phenolic content for both litchi varieties. Our result is inagreement with Soong and Barlow (2004) and Othman, Ismail, Ab-dul Ghani, and Adenan (2007) who also found a strong correlationbetween the total phenolic content and the FRAP values. It isknown that phenolic compounds exhibit redox properties (i.e.,act as reducing agents, hydrogen donators and singlet oxygenquenchers). This property of phenol may account for the strong po-sitive correlation observed between FRAP and total phenolic con-tent for both litchi varieties. Furthermore, both antioxidantassays, FRAP and DPPH scavenging, were found to be positivelycorrelated; their correlation values were 0.9 and 0.73, for ‘Shahi’and ‘China’, respectively. This may be attributed to the major con-tribution of phenolics in both litchi varieties as evident from thecorrelation matrix (Table S1A and S1B).

Compared to phenolics, a weaker correlation was observed be-tween vitamin C and the antioxidant property of ‘Shahi’(Table S1A). However, a positive correlation was found for the ‘Chi-na’ variety (Table S1B). As already mentioned elsewhere, the litchifruit of ‘China’ variety was found to have a significantly higher vita-min C content compared to ‘Shahi’. The contribution of vitamin Ctowards the total antioxidant content is only about 2% for plumsas reported by Rupasinghe, Jayasankar, and Lay (2006). They alsopointed out that non-vitamin C phytochemicals, such as polyphe-nols may be playing a crucial role in the antioxidant value. It seemsthat the relationship between antioxidant activity and phyto-chemicals depends on several factors, such as the characteristicsof individual phytochemicals and the synergistic interactionamong them.

4. Conclusion

Commercially grown Indian litchi cultivars ‘Shahi’ and ‘China’were found to be rich sources of antioxidants and possessed strongradioprotective ability, as evidenced by the significant protectiontoward the pBR322 plasmid DNA, even at a dose of 5 kGy. Thiswas further confirmed by the protection of E. coli cells by the litchijuice. Neither radiation processing nor low temperature storage ofjuice affected these properties.

Acknowledgements

The authors wish to express their gratitude to Ms. Ujjwala Tri-pathy and Ms. Soniya Shah for their technical help.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.foodchem.2010.10.051.

References

ASTM. (1993). E1205-93. Standard practice for use of a ceric-cerous dosimetry system.Annual book of standards (Vol. 12.02). 100 Barr Harbor Drive, WestConshohoken, PA 19428, USA: ASTM.

Chen, H. Y., Lin, Y. C., & Hsieh, C. L. (2007). Evaluation of antioxidant activity ofaqueous extract of some selected nutraceutical herbs. Food Chemistry, 104(4),1418–1424.

Clifford, M. N. (1995). Understanding the biological effects of dietary complex phenolsand tannins and their implications for the consumer’s health and well being. Reportof the European project FAIR-CT95-0653. European Community Programme forResearch, Technological Development and Demonstration in the field ofAgriculture and Fisheries.

Connor, A. M., Luby, J., Hancock, J. F., Berkheimer, S., & Hanson, E. J. (2002). Changesin fruit antioxidant activity among blueberry cultivars during cold-temperaturestorage. Journal of Agricultural and Food Chemistry, 50, 893–898.

Dani, M., Nair, C. K. K., Jacob, A. J., Biswas, N., Mukherjee, T., & Kapoor, S. (2007).Ascorbic acid monoglucoside as antioxidant and radioprotector. Journal ofRadiation Research, 48, 369–376.

Dragaric, I. G., & Dragaric, Z. D. (1971). The radiation chemistry of water p. 256. NewYork: Academic Press.

Hajare, S. N., Saxena, S., Kumar, S., Wadhawan, S., More, V., Mishra, B. B., et al.(2010). Quality profile of litchi (Litchi chinensis) cultivars from India and effectof radiation processing. Radiation Physics and Chemistry, 79(9), 994–1004.

Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review ofNutrition, 16, 33–50.

Häussler, S., & Becker, T. (2008). The Pseudomonas Quinolone Signal (PQS) balanceslife and death in Pseudomonas aeruginosa populations. PLoS Pathogens, 4(9), 1–8.

Heather, N. W., & Hallman, G. J. (2008). Phytosanitation with ionizing radiation. InN. W. Heather & G. J. Hallman (Eds.), Pest management and phytosanitary tradebarriers (pp. 132–152). Wallingford, UK: CAB International.

Henson, D. E., Block, G., & Levine, M. (1991). Ascorbic acid: Biologic functions andrelation to cancer. Journal of the National Cancer Institute, 83, 547–550.

Kada, T., Kaneko, K., Matsuzaki, S., & Hara, Y. (1985). Detection and chemicalidentification of natural bioantimutagens. A case of green tea factor. MutationResearch, 150, 127–132.

Kevers, C., Falkowski, M., Tabart, J., Defraigne, J.-O., Donimes, J., & Pincemail, J.(2007). Evolution of antioxidant capacity during storage of selected fruits andvegetables. Journal of Agricultural and Food Chemistry, 55, 8596–8603.

S. Saxena et al. / Food Chemistry 126 (2011) 39–45 45

Lee, H. S., & Wicker, L. (1991). Anthocyanin pigments in the skin of lychee fruit.Journal of Food Science, 56, 466–483.

Liu, R. H. (2003). Health benefits of fruits and vegetables are from additive andsynergistic combination of phytochemicals. American Journal of ClinicalNutrition, 78, 517S–520S.

Luximon-Ramma, A., Bahorun, T., & Crozier, A. (2003). Antioxidant actions andphenolic and vitamin C contents of common Mauritian exotic fruits. Journal ofthe Science of Food and Agriculture, 83, 496–502.

Othman, A., Ismail, A., Abdul Ghani, N., & Adenan, I. (2007). Antioxidant capacityand phenolic content of cocoa beans. Food Chemistry, 100, 1523–1530.

Oyaizu, M. (1986). Antioxidative activity of browning products of glucosaminefractionated by organic solvent and thin-layer chromatography. NipponShokuhin Kogyo Gakkaishi, 35, 771–775.

Patrineli, A., Clifford, M. N., Walker, R., & Ioannides, C. (1996). Mutagenicity of whitegrape juice in the Ames test. Food and Chemical Toxicology, 34, 559–562.

Pradhan, D. S., Nair, C. K. K., & Sreenivasan, A. (1973). Radiation injury repair andsensitization of microorganisms. Proceedings of the Indian National ScienceAcademy, 39B, 516–530.

Prasad, N. K., Yang, B., Ruenroengklin, N., Zhao, M., & Jiang, Y. (2009). Application ofultrasonication or high pressure to assist extraction of flavonoids from litchifruit pericarp. Journal of Food Process Engineering, 32, 828–843.

Prasad, N. K., Yang, B., Yang, S., Chen, Y., Zhao, M., Ashraf, M., et al. (2009).Identification of phenolic compounds and appraisal of antioxidant andantityrosinase activity of litchi (Litchi sinensis Sonn.) seeds. Food Chemistry,116, 1–7.

Rice-Evans, C., Miller, N. J., & Paganga, G. (1996). Structure–antioxidant activityrelationships of flavonoids and phenolic acids. Free Radical Biology and Medicine,20, 933–956.

Rivera-López, J., Ordorica-Falomir, C., & Wesche-Ebeling, P. (1999). Changes inanthocyanin concentration in Lychee (Litchi chinensis Sonn.) pericarp duringmaturation. Food Chemistry, 65, 195–200.

Robards, K., Prenzler, P. D., Tucker, G., Swatsitang, P., & Glover, W. (1999). Phenoliccompounds and their roles in oxidative process in fruits. Food Chemistry, 66,401–436.

Rodriguez-Saona, L. E., Fry, F. S., McLaughlin, M. A., & Calvey, E. M. (2001). Rapidanalysis of sugars in fruit juices by FT-NIR spectroscopy. Carbohydrate Research,336, 63–74.

Rupasinghe, H. P. V., Jayasankar, S., & Lay, W. (2006). Variation in total phenolicsand antioxidant capacity among European plum genotypes. ScientiaHorticulturae, 108, 243–246.

Salunkhe, D. K., & Desai, B. (1984). Postharvest biotechnology of fruits pp. 77–80.Florida: CRC Press.

Sharma, A., Gautam, S., & Jadhav, S. S. (2000). Spice extracts as dose-modifyingfactors in radiation inactivation of bacteria. Journal of Agricultural and FoodChemistry, 48, 1340–1344.

Shivashankara, K. S., Isobe, S., Al-Haq, M. I., Takenaka, M., & Shina, T. (2004). Fruitantioxidant activity, ascorbic acid, total phenol, quercitin, and carotene of Irwinmango fruits stored at low-temperature after high electricfield treatment.Journal of Agricultural and Food Chemistry, 52, 1281–1286.

Singh, S. P., & Pal, R. K. (2009). Ionizing radiation treatment to improve postharvestlife and maintain quality of fresh guava fruit. Radiation Physics and Chemistry,78, 135–140.

Singleton, V. L., Orthofer, R., & Lamuela-Raventos, R. M. (1999). Analysis of totalphenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Methods in Enzymology, 299, 265–275.

Soong, Y. Y., & Barlow, P. J. (2004). Antioxidant activity and phenolic content ofselected fruit seeds. Food Chemistry, 88, 411–417.

Tavarini, S., Degl’Innocenti, E., Remorini, D., Massai, R., & Guidi, L. (2008).Antioxidant capacity, ascorbic acid, total phenols and carotenoids changesduring harvest and after storage of Hayward kiwifruit. Food Chemistry, 107,282–288.

Temple, N. J. (2000). Antioxidants and disease: More questions than answers.Nutrition Research, 20, 449–459.

Zhang, P., & Omaye, S. T. (2001). DNA strand breaking and oxygen tension: Effects ofb-carotene, a-tocopherol and ascorbic acid. Food and Chemical Toxicology, 39,239–246.