Contents lists available at SciVerse ScienceDirect
Industrial Crops and Products
journa l h o me pag e: www.elsev ier .com/ locate / indcrop
apid separation of carotenes and evaluation of their in vitro antioxidantroperties from ripened fruit waste of Areca catechu – A plantation crop ofgro-industrial importance
atural Product Biotechnology Group, Agricultural & Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721 302, India
r t i c l e i n f o
rticle history:eceived 17 December 2011eceived in revised form 12 March 2012ccepted 14 March 2012
ey words:reca catechu-Carotene
a b s t r a c t
The waste product generated during arecanut processing is the ripened pericarp. The ripened peri-carp tissue of Areca catechu accumulates carotene compounds, nevertheless remained unexplored asa source of tapping colourants. A reverse phase-thin layer chromatography (RP-TLC)-based separationwas standardised for the first time for pericarp carotenes in A. catechu. UV–vis spectroscopy formed thebasis for identification of major pericarp carotenes, and the method was validated subsequently. Solidphase extraction was also performed for rapid separation of �-carotene, using Sep-Pak® cartridge. It wasfound that �-carotene constitutes ∼30% of the total carotenoid content in the pericarp tissues. The total
P-TLCep-Pak® cartridgentioxidant activity
carotenoid content was found to be 11.67 ± 0.62 mg �-carotene equivalent per 100 g fresh mass of peri-carp tissue. The antioxidant capacities of the extractives were evaluate by four different commonly usedin vitro assays namely, ferrous reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH),and 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and DNA nicking assay. Results of allthe antioxidant assays point out a significant antioxidant property of the agro-industrial fruit waste of A.catechu.
. Introduction
Areca catechu is an important cash crop belongs to Arecaceaeamily and widely cultivated in tropical and subtropical regionsor harvesting arecanut. Arecanut (seed of A. catechu) colloquiallynown as “Supari”, has a long history of consumption as a masti-ator item in India and other countries (Peter, 2002). The pericarpissue constitutes 15–30% of the raw nut. These tissues are sepa-ated out from the ripened fruit during arecanut processing anderve mostly as wastes (Peter, 2002). Arecaceae fruit wastes asuch contain a rich source of phytochemicals (Chakraborty et al.,006), and phenolic compounds isolated from the mesocarp wastef another Arecaceae species Cocos nucifera, exhibited promisingntioxidant properties (Chakraborty and Mitra, 2008). Thus, fruitastes of Arecaceae have the potential to serve as a sustainable
ource of bioactive compounds.The agro-industrial fruit pericarp wastes taken out from ripened
ruit of A. catechu are orange-red in colour. During the process ofipening, a decrease in the content of chlorophyll and a massiveoncomitant accumulation of carotenes are evident as observed
in different cultivars of a palm fruits including Elaeis germansis(Ikemefuna and Adamson, 1984). Therefore, it can be assumed thatthe carotenoids accumulated in the ripened fruit of A. catechu areresponsible for the orange-red colour of the pericarp. Carotenesare strong antioxidants and have been credited with many ben-eficial effects on human health. Carotenoids have beneficial rolein the prevention of several chronic diseases like cardiovasculardiseases and cancer (Giovannucci et al., 2002). Carotenes are alsobeing used for a long time as food colourants. Uses of A. catechupericarp as a source for tapping colourants are apparently unex-plored (Peter, 2002). Limited information is available on the profilesof phenolic compounds in A. catechu pericarp (Chakraborty et al.,2006) and surprisingly, no information is available on the natureof carotene compounds that accumulate in the pericarp tissue ofA. catechu.
India produces 0.476 million tonnes of arecanut annually, dom-inates the world in terms of productivity (FAO, 2008). As a resulthuge amount of A. catechu pericarp wastes are generated everyyear with almost no attention being given to utilise these biologi-cal wastes. The work described in this report is the first systematicattempt to explore this agro-industrial fruit waste for harnessing
carotenes. Here we report a rapid and simple technique for isolationand analysis of carotenes from A. catechu pericarp. The antioxidantcapacities of the extractives were also evaluated in vitro to emphasistheir beneficial roles in human health.
Fresh and ripened A. catechu fruits were collected from localrchard in Hooghly district, West Bengal and brought to Kharagpur.ericarp tissues of the ripened fruit were separated out from the nutnd subsequently used for extraction of carotene compounds. Allhe analytical grade chemicals used in this study were purchasedrom E. Merck (India), Sigma–Aldrich (USA) and Himedia (India).tandard �-carotene was purchased from Himedia (India). All com-ercially available TLC plates (cellulose, silica gel 60 F254 and RP-18
ilica F254) were purchased from Merck (Darmstadt, Germany). C-8 Sep-Pak® cartridge, used for solid phase extraction procuredrom Waters (Milford, USA).
.2. Sample preparation
A defined amount (∼30 g) of fresh pericarp tissues of wasxtracted with 100 ml of acetone (100%) in a Soxhlet apparatus at5 ◦C for 12 h. The acetone extract was subsequently partitionedith double volume of heptane in a separating funnel. Upper hep-
ane layer was evaporated to dryness in rotary vacuum evaporatort 30 ◦C. It was later resuspended in methanol (100%, v/v) and storedn dark at 4 ◦C, which was subsequently used in TLC and SPE. For
easuring antioxidant activity the dried sample was resuspendedn ethanol (95%, v/v).
.3. Standardisation of TLC separation
A series of TLC analyses were performed with three differenttationary phases (using aluminium sheeted commercial plates)amely, silica gel 60 F254, cellulose and RP-18 silica F254 using aumber of mobile phases including methanol:diethyl ether:water56:40:5). TLC plates were viewed under white light.
.4. Solid phase extraction for rapid isolation of ˇ-carotene
Solid phase extraction was performed with C-18 Sep-Pak® car-ridge. The mobile phase used was a mixture of methanol, diethylther and water in a ratio of 56:40:5. Sample was prepared by com-letely evaporating the methanolic extract in rotary evaporator andesuspended in 1.5 ml of the said mobile phase. The cartridge wasquilibrated with the mobile phase twice, using injection syringeefore loading the sample of 1.5 ml volume. This was followed by
njection of mobile phase with a flow rate maintained at 1 ml/min.ltogether, 24 fractions (each with 0.5 ml volume) collected in thependorf tube were subsequently subjected to spectral analysis inhe wavelength range of 300–600 nm. Fractions showing identi-al spectrum were pooled together and partially concentrated inotary vacuum evaporator. Thereafter two phase separation waserformed using ethyl acetate. Upon formation of two layers, upper
ayer was taken out and completely evaporated to dryness in rotaryaccum evaporator and later resuspended in methanol (100%, v/v)or TLC analysis and quantification.
.5. Characterization of carotenes
Both TLC-separated and Sep-Pak® cartridge-separated caroteneractions were subjected to spectral analysis for characterization.pectral scanning of the separated carotenes was carried out in aV-1800 UV–vis Spectrophotometer (Shimadzu, Japan) between
00 and 600 nm.
For analyses of carotenes, TLC was performed on RP-18 silica254 silica commercial plate of convenient size (5 cm × 7.5 cm size).ample was spotted in band mode. The mobile phase used for
Products 40 (2012) 204– 209 205
TLC run was same of that of used in solid phase extraction. TLCseparated bands and Sep-Pak® cartridge separated fractions weresubsequently subjected to spectral analysis.
2.6. Quantification of total carotene and ˇ-carotene
The spectrophotometric determination of total carotene wascarried out as described before (Jansen, 1978). The total carotene inthe sample was determined by �-carotene standard and the con-centration of total carotene content was expressed as �-caroteneequivalent. Quantification of �-carotene was also done by samemethod described above. The methanolic fraction of scraped bandof �-carotene separated from TLC and the methanolic fraction of�-carotene separated from Sep-Pak® cartridge were subjected toquantification.
2.7. Validation of TLC method for quantification of ˇ-carotene
The TLC method established for separation and quantificationof �-carotene from pericarp tissues of A. catechu fruits was vali-dated for specificity, repeatability and accuracy in accordance withICH guidelines. The identity of the �-carotene band was confirmedby comparing both Rf value and spectral property with that of �-carotene standard. The repeatability of the method was confirmedby repeated analyses (six times) of the sample with spot quantity3.94 �g (in band mode) and subsequent expression as coefficientof variance (% RSD). The accuracy of the method was tested by per-forming recovery studies of the pre-analysed sample at two levels(100% level and 50% level), and percentage recovery was calculated.For recovery studies, known amount of standard �-carotene (4.5 �gand 2.25 �g for 100% and 50% levels, respectively) was added in thesample solution containing 4.5 �g of �-carotene and spotted in theRP-TLC plate for TLC analyses.
2.8. Antioxidant properties
2.8.1. DPPH free radical scavenging assayThe scavenging activity was estimated using DPPH as free radi-
cal as reported earlier (Brand-Williams et al., 1995). The ethanolicextract of total carotene and �-carotene (separated by SPE) wasmonitored for scavenging activity. An aliquot of ethanol solu-tion (0.1 ml) containing different concentrations of extract (totalcarotenoid extract and �-carotene extract) was mixed with 0.9 mlethanolic solution of DPPH (0.1 mM). The mixture was shaken vig-orously and left to stand for 30 min at room temperature in the dark.The decrease in the absorbance was monitored at 517 nm. Ascorbicacid was taken as a standard antioxidant. The result was expressedas IC50 (50% inhibitory concentration). Inhibition of free radicals byDPPH in percent (I%) was calculated using following formula:
I% =(
Acontrol − Asample
Acontrol
)× 100
where Acontrol is the absorbance of the control reaction (contain-ing all reagents except the test compound), and Asample is theabsorbance of the test compound. From I% values IC50 was calcu-lated.
2.8.2. ABTS free radical scavenging assayThis assay was performed according to a published method (Re
et al., 1999). Briefly, the pre-formed ABTS free radicals were gener-ated by reacting ABTS solution (7 mM) with 2.45 mM potassium
persulfate and keeping it for 16 h in dark at room temperature.The solution was diluted with ethanol (95%, v/v) till getting theabsorbance 0.7 ± 0.2 units at 734 nm. The ethanolic sample (totalcarotenoid extract) of 20 �l (concentration 107.6 �g/ml) was mixed
206 M. Kumar et al. / Industrial Crops and Products 40 (2012) 204– 209
Fig. 1. RP-TLC separation of carotenes and spectral overlays. (a) Separation of carotene compound in RP-TLC plate using methanol:diethyl ether:water (56:40:5) as mobilep s wers that ot
wswtccw
2
i(pialt3tf
2a
ao
hase. The carotenes were extracted from ripened pericarp of A. catechu. These bandcan between 300 and 600 nm. (b) Spectral overlay of band 1 (from the base) withhe base), advocating identical spectral nature and �max values.
ith 1 ml of ABTS free radical solution. The absorbance was mea-ured spectrophotometrically at 734 nm after 6 min. All reactionsere performed in triplicate. The free radical scavenging activity of
he extract was expressed as ascorbic acid equivalent antioxidantapacity (AEAC), which was obtained by comparing absorbancehange at 734 nm in a reaction mixture containing the test sampleith that of calibration plot of ascorbic acid for ABTS assay.
.8.3. Reducing power assayThe reducing power was determined by ferric reducing antiox-
dant power (FRAP) assay as described by Benzie and Strain1996) with some modification. FRAP reagent was freshly pre-ared by mixing TPTZ (2,4,6-tripyridyl-s-triazine) solution (10 mM
n 40 mM HCl), ferric chloride solution (20 mM in distilled water)nd acetate buffer (300 mM, pH 3.6) in a ratio of 1:1:10. The ethano-ic carotenoid extract (100 �l, concentration 107 �g/ml) was addedo 2.9 ml of FRAP solution and kept for incubation for 6 min at7 ◦C. The absorbance was recorded at 593 nm. Reducing poten-ial was expressed in units of mmol Fe2+/g of pericarp tissue (onresh weight basis).
.8.4. DNA nicking assay for non site specific •OH scavenging
ctivity
This assay was performed according to Kitts et al. (2000) with slight modification. The nicking reaction mixture contained 6 �lf pUC18 plasmid in 4 �l of KH2PO4 buffer (50 mM, pH 7.4), 4 �l
e later scraped (as described in materials and methods) for recording their spectralf �-carotene standard. (c) Spectral overlay of bands 2–4 (in ascending order from
of total carotenoid extract (0.1 mg/ml), 4 �l of EDTA-Na2 (30 mM),4 �l of H2O2 (30 mM) and 4 �l of FeSO4 (16 mM). The reactionmixture volume was adjusted to 26 �l using deionized distilledwater. The reaction mixture was incubated for 1 h at 37 ◦C. Afterincubation, 4 �l of 6× loading dye (fermentas) was added and thesamples were then analysed by electrophoresis on 1.2% agarosehorizontal slab gel with 3.5 �l of ethidium bromide (10 mg/ml) intris-acetate EDTA-Na2 buffer (40 mM tris-acetate and 1 mM EDTA,pH 8.4) at 80 V. DNA bands were then visualised and subsequentlyphotographed using a gel documentation system (DNR Bio-ImagingSystems).
3. Results and discussion
3.1. Carotenes separation by RP-TLC and solid phase extraction
Thin layer chromatography on silica gel layer continues toremain a simple and useful way of carrying out separation ofdifferent carotene compounds. In this work, a range of differentstationary phases were used such as, cellulose, silica G-60 F254 andRP-18 silica F254. TLC chromatograms obtained during standard-isation of separation using various stationary and mobile phases
demonstrated that the separation on RP-18 silica F254 TLC platewith mobile phase consisting of methanol, diethyl ether and waterin the ratio of 56:40:5 gave the best result (Fig. 1). Eight distinctbands were visible in this case as compared to mobile ratios of
M. Kumar et al. / Industrial Crops and Products 40 (2012) 204– 209 207
Fig. 2. Isolation of �-carotene by solid phase extraction. (a) Spectral overlay of Sep-Pak® cartridge-separated fractions (fractions 8–16) with that of standard �-carotene.The deep red colour spectrum indicates the standard �-carotene. (b) TLC chromatogram of crude carotene (lane 1), Sep-Pak® cartridge-separated carotenes pooled fromfractions 8–17 (lane 2) and fractions 4–6 (lane 3). The figure clearly indicates �-carotene separation in SPE, collected in 9 consecutive fractions (8–16). All other carotenes( the ret
5twiT(Fmro
ctcsosg�cuvlu(tbscw(ti
mwroAec
except �-carotene) are eluted between serial numbers 4–6. (For interpretation of
he article.)
6:40:8 and 56:40:4 where four and eight bands (not very dis-inct), were found to be resolved, respectively, when RP-TLC platesere used (figure not shown). Similar observation was also found
n Sorbus aucuparia berries, where carotene separation on RP-18LC plate was found to be satisfactory over normal phase systemsAndersan and Francis, 2004). However, when cellulose and silica254 was used as matrix with methanol, diethyl ether and water asobile phase individually, only two bands were found to be sepa-
ated in both cases; use of acetone as mobile phase only resolvedne band in cellulose plate.
UV–vis spectrum obtained for band 1 (Fig. 1a) showed identi-al spectral property with the spectrum of the �-carotene (Fig. 1b),hus suggested it to be �-carotene and was further confirmed byomparing Rf with the standard. The similarities observed on thepectral overlay for bands 2–4 (Fig. 1c) was indicative of isomersf a single carotene. Similarly, appearance of similar patterns inpectral overlay for bands 5 and 6 (spectrum not shown) also sug-ested to be isomers of a yet to be identified carotene. Apart from-carotene, no other carotene standards were available commer-ially. But this limitation did not hinder the characterization ofnknown carotenoids. A comparison of the absorption maximaalues of the generated spectra with that of data available in theiterature on the absorption maxima of different carotenes alloweds to identify some of these carotenes compounds, tentativelyIttah et al., 1993). Using such approach, bands 2–4 were suggestedo be isomers of �-carotene as the absorption maxima of theseands matched very well with the available data for �-carotenepectrum (Breitenbach and Sandmann, 2005). Accumulation of �-arotene is plausible in A. catechu pericarp like yellow passion fruit,here accumulation of such carotene has already been reported
Mercadante et al., 1998). Nevertheless, high-resolution mass spec-rometry will be required for chemical confirmation of �-carotenen A. catechu pericarp.
Calibration curve was plotted for �-carotene standard for deter-ining total carotenoid content and �-carotene content. Linearityas found between the concentration range of 1 and 50 �g with
2 = 0.981 ± 0.005. Linearity was evaluated by taking absorbance
f eleven different concentrations of �-carotene in triplicates.bsorbance and concentration were subjected to least square lin-ar regression analysis for calculating the calibration equation andorrelation coefficient.
ferences to colour in this figure legend, the reader is referred to the web version of
In order to isolate �-carotene from crude carotene mixtures,solid phase extraction method was devised using commerciallyavailable matrix. The crude extract was loaded onto a Sep-Pak®
cartridge and subsequently eluted with the same mobile whichgave best resolution of separated carotenes in RP-TLC plate. Theelutes were analysed by UV–vis spectroscopy (Fig. 2a) followed byRP-TLC analysis standardised before (Fig. 2b). The total carotenoidcontent was found to be 11.67 ± 0.62 mg �-carotene equivalent per100 g fresh mass of pericarp tissue while the �-carotene contentwere 3.32 ± 0.07 mg/100 g of fresh pericarp tissue when separationis done by RP-TLC. The �-carotene value obtained with solid phaseextraction method was 3.64 ± 0.02 mg/100 g of fresh mass of peri-carp. A comparison of the quantity of �-carotene accumulation inthe pericarp tissue isolated through Sep-Pak® cartridge with thatof TLC, showed essentially identical results. Thus the separation of�-carotene using Sep-Pak® cartridge appeared to be not only rapidbut also very promising in terms of recovery. Also, suggesting totalcarotenoid content and �-carotene content determined in pericarptissues of ripened A. catechu fruit are within the range of carotenecontents found in different fruits and vegetables (Rajyalakshmiet al., 2003).
3.2. Validation of RP-TLC method for quantification of ˇ-carotene
The TLC method standardised here was also validated for speci-ficity, repeatability and accuracy. The established method wasfound to be specific for �-carotene with good resolution. Thespectral scans of RP-TLC elute and SPE elute matched very wellwith the standard �-carotene (Figs. 1b and 2a). Moreover, RP-TLCchromatogram showed identical Rf (0.105) values. The repeata-bility result of �-carotene was expressed in RSD and the valuedetermined was 0.8, suggesting that no significant variation wasobserved in the repeatability analysis of �-carotene. The RSD valuewas less than 2%, suggested that the method is reproducible. Therecovery studies were done by comparing the concentration foundin the samples spiked with known amount (100% and 50%) of �-carotene and by performing TLC analyses before and after addition.
The recovery (mean ± SD) was 99.41 ± 1.49%, thereby advocatingthe method to be accurate.
Different normal and RP TLC methods are available for separa-tion of plant carotenoids (Isaksen and Francis, 1986; Andersan and
208 M. Kumar et al. / Industrial Crops and Products 40 (2012) 204– 209
Table 1Total carotenoid and �-carotene content of fruit pericarp waste of A. catechu and their antioxidant activity, as well as DPPH value (in terms of IC50) of ascorbic acid standard.
rancis, 2004), none of the methods worked as efficient as the RPLC method used here for the separation of carotenes from A. cate-hu pericarp. In recent years ultrasonic treatment couple with HPLCnalysis (Sun et al., 2011) and HPLC–DAD–MS analytical methodsor characterization of carotenes are available (Fraser et al., 2000;entili and Caretti, 2011). But these require expensive equipmentshich often limits its widespread use in developing countries.
.3. Antioxidant capacities
The antioxidant capacities of A. catechu pericarp tissues weressessed in vitro by four different chemical assays: DPPH, ABTS,RAP and DNA nicking assay.
.4. Determination of DPPH free radical scavenging activity
DPPH, a stable free radical with a characteristic absorptiont 517 nm was used to study the radical scavenging effects ofarotenoids extracted in ethanol from pericarp tissues of ripenedreca fruit. Decreases in absorbance are observed when antiox-dants donate protons to these free radicals. This decrement inbsorbance is taken as a measure of the extent of radical scavenging.
The quantitative result shown in Fig. 3a indicated that-carotene showed a higher anti-radical capacity than totalarotenoid extract at very low concentration. However, whileeaching towards IC50 value, it was observed that the scavengingctivity of both �-carotene and total carotenoid extract appeareddentical. This phenomenon could possibly be explained in theollowing way. The �-carotene content in the total carotenoidxtract as compared to the �-carotene extract was less. How-ver, the total carotenoid extract consisted not only of �-carotene,
ut also �-carotene as well as the presence of other unidenti-ed carotenes. It appeared that the synergistic effect of differentarotenes present in the total carotenoid extract was not very effec-ive at a lower concentration. However, when the concentration
ig. 3. DPPH free radical scavenging activity of total carotenoid extract, �-carotenextract and ascorbic acid standard.
2.07 ± 0.07 –
of total carotenoid extract was increased, the synergistic effect ofdifferent carotenes came into action (Palozza and Krinsky, 1991).This is also the reason for the high scavenging activity of totalcarotenoid extract as compared to �-carotene extract alone, asshown in Fig. 3a. The result also showed that there is no apprecia-ble increase in DPPH inhibition despite increasing concentrationof �-carotene. The antioxidant activity of �-carotene is due to itsconjugated polyene carbon centre which interacts with the freeradicals. Due to being a bicyclic molecule, a hindrance is gener-ated between polyene carbon centre of �-carotene and DPPH freeradicals might have caused less substantial increase in antioxidantactivity despite of increase in concentration (Mueller and Boehm,2011). When the total carotenoid extract, �-carotene extract (sep-arated by SPE) and ascorbic acid were compared, it was found thatascorbic acid standard having best DPPH free radicals scaveng-ing activity among three with IC50 value 2.07 ± 0.07 �g/ml whiletotal carotenoid extract showed better anti-radical activity than�-carotene alone. The IC50 value for total carotenoid extract and �-carotene were found to be 7.92 ± 0.08 �g/ml and 6.29 ± 0.01 �g/ml,respectively.
3.4.1. ABTS free radical scavenging assayThis assay was done with total carotenoid extract of ripened
areca pericarp only and the value was represented as AEAC (ascor-bic acid equivalent antioxidant capacity). The ABTS value was foundto be 1.03 ± 0.01 �M/�g AEAC (Table 1).
3.4.2. The reducing power assayFRAP assay was done to evaluate the reducing capacity of total
carotenoid extract of areca pericarp. In FRAP assay, the antioxi-dants in the sample were treated as reductant in a redox-linkedcolorimetric reaction (Guo et al., 2003). The antioxidants reducethe ferric tripyridyltriazine complex to its blue coloured ferrousform (Benzie and Strain, 1996), which can be detected at 593 nm.This assay is simple and easy to standardise, hence, it has beenused frequently in the assessment of antioxidant activities of var-ious fruits and vegetables (Guo et al., 2003). The FRAP values forthe total carotenoid extract and �-carotene extract of A. catechupericarp fruit waste were 4.23 ± 0.22 and 0.44 ± 0.005 mM Fe2+/gof fresh pericarp, respectively (Table 1).
3.4.3. DNA nicking assayThis study was performed to evaluate the protective activity of
total carotenoid extract of A. catechu against the Fenton induced•OH damage of DNA. The pUC18 plasmid DNA was used as thesource of DNA. Most of the oxidative damage in biological system iscaused by the •OH which is generated by the reaction between O2
•−
and H2O2 in the presence of metal ions. The Fenton agents causenon site-specific cleavage of supercoiled form of DNA (Fig. 4). The
result showed (lane B). This damage was found to be prevented inDNA treated with Fenton’s agents in presence of total carotenoidextract (0.1 mg/ml) of A. catechu (lane C) as it showed almost similarpattern with that of the control (lane A).
M. Kumar et al. / Industrial Crops and
Fig. 4. Agarose gel electrophoretic pattern of Fenton induced •OH damage ofDeD
4
totssmianp
A
MhTfpf
R
A
NA using pUC18 plasmid DNA in the presence and absence of total carotenoidxtract of A. catechu Lane A, control DNA; Lane B, DNA + Fenton’s reagent; Lane C,NA + Fenton’s reagent + total carotenoid areca extract (0.1 mg/ml).
. Conclusion
The RP-TLC and SPE methods described here are simple, selec-ive, precise and accurate means of separation and quantificationf �-carotene in ripened pericarp of A. catechu. The quantifica-ion analysis advocate that ripened pericarp of A. catechu is a goodource for harnessing carotenes especially �-carotene. Sensitivityimplicity and rapidity are the main advantages of this method. Theethod can be used in routine analysis for separation of carotenes
ncluding �-carotene from plant sources. Moreover, bioactivitynalysis of the ripened pericarp extract A. catechu showed sig-ificant antioxidant property, making this fruit agro-waste as aotential candidate for natural antioxidants.
cknowledgements
This communication represents part of M. Tech. thesis work ofahesh Kumar, who was a recipient of GATE scholarship to pursue
is M. Tech. programme in Applied Botany at the Indian Institute ofechnology Kharagpur. Utkarsh Ravindra Moon thanks UGC, Indiaor the award of an individual junior research fellowship. It is aleasure to thank Chiranjit Mukherjee for collecting A. catechu fruitsor this work.
eferences
ndersan, O.M., Francis, G.W., 2004. Techniques of pigment identification. In: PlantPigment and their Manipulation. Blackwell/CRC Press, Oxford, UK, pp. 293–341.
Products 40 (2012) 204– 209 209
Benzie, I.E.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP)as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239,70–76.
Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method toevaluate antioxidant activity. Food Sci. Technol. 28, 25–30.
Breitenbach, J., Sandmann, G., 2005. �-Carotene cis isomers as products and sub-strates in the plant poly-cis carotenoid biosynthetic pathway to lycopene. Planta220, 785–793.
Chakraborty, M., Das, K., Dey, G., Mitra, A., 2006. Unusually-high quantity of 4-hydroxybenzoic acid accumulation in cell wall of palm mesocarps. Biochem.Syst. Ecol. 34, 509–513.
Chakraborty, M., Mitra, A., 2008. The antioxidant and antimicrobial propertiesof the methanolic extract from Cocos nucifera mesocarp. Food Chem. 107,994–999.
performance liquid chromatography with photodiode array detection to themetabolic profiling of plant isoprenoids. Plant J. 24, 551–558.
Gentili, A., Caretti, F., 2011. Evaluation of a method based on liquidchromatography–diode array detector–tandem mass spectrometry fora rapid and comprehensive characterization of the fat-soluble vitaminand carotenoid profile of selected plant foods. J. Chromatogr. A 1218,684–697.
Giovannucci, E., Rimm, E.B., Liu, Y., 2002. A prospective study of tomato productslycopene, and prostate risk. J. Natl. Cancer Inst. 94, 391–398.
Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Jiang, Y., 2003. Antioxidant activities of peel pulpand seed fractions of common fruits as determined by FRAP assay. Nutr. Rev. 23,1719–1726.
Ikemefuna, J., Adamson, I., 1984. Chlorophyll and carotenoid changes in ripeningpalm fruit, Elaeis guineënsis. Phytochemistry 23, 1413–1415.
Isaksen, M., Francis, G., 1986. Reversed-phase thin layer chromatography ofcarotenoids. J. Chromatogr. 355, 358–362.
Ittah, Y., Kanner, J., Granit, R., 1993. Hydrolysis study of carotenoid pigmentsof paprika by HPLC/photodiode array detection. J. Agric. Food Chem. 41,899–901.
Jansen, A., 1978. Handbook of Phycological Methods: Physiological and BiochemicalMethod. Cambridge University Press, Cambridge, pp. 59–70.
Kitts, D.D., Wijewickreme, A.N., Hu, C., 2000. Antioxidant properties of a NorthAmerican ginseng extract. Mol. Cell. Biochem. 203, 1–10.
Mercadante, A.Z., Britton, G., Rodriguez-Amaya, D.B., 1998. Carotenoid from yellowpassion fruit (Passiflora edulis). J. Agric. Food Chem. 46, 4102–4106.
Mueller, L., Boehm, V., 2011. Antioxidant activity of �-carotene compounds in dif-ferent in vitro assays. Molecules 16, 1055–1069.
Palozza, P., Krinsky, N.I., 1991. The inhibition of radical-initiated peroxidation ofmicrosomal lipids by both �-tocopherol and �-carotene. Free Radic. Biol. Med.11, 407–414.
Peter, K.V., 2002. Arecanut. In: Plantation Crops. National Book Trust, New Delhi, pp.1–28.
Rajyalakshmi, P., Padmavathi, T.V.N., Venkata Laxmi, K., 2003. Total carotenoid and�-carotene contents of other vegetables roots and tubers, fruits and stored prod-ucts collected from forest areas of Andhra Pradesh, South India. Plant Foods Hum.Nutr. 58, 1–11.
Re, R., Pellegrinni, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999.
Sun, Y., Liu, D., Chen, J., Ye, X., Yu, D., 2011. Effects of different factors of ultrasoundtreatment on the extraction yield of the all-trans-b-carotene from citrus peels.Ultrason. Sonochem. 18, 243–249.
