Research ArticleReceived: 19 September 2012 Revised: 5 January 2013 Accepted article published: 29 January 2013 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6081

Separation of polyphenols and arecoline fromareca nut (Areca catechu L.) by solventextraction, its antioxidant activity, andidentification of polyphenolsYogita V Chavan and Rekha S Singhal

Abstract

BACKGROUND:Arecanut (Arecacatechu L.) or betel nut, a commercial cash crop, is a rich sourceof polyphenols but also containstoxic alkaloids, mainly arecoline. Separation of these bioactive polyphenols from toxic constituents could propel the safe andbeneficial use of betel nut; also it will help arecanut processing industries to produce arecoline-free products. With the aim todevelop an effective method for maximum extraction of polyphenols with minimum arecoline, several factors such as natureof the solvent, pH (210), substrate concentration (614 %) and extraction time (30150 min) under shaking conditions wereevaluated. Qualitative analysis was done using spectrophotometry and high-performance liquid chromatography (HPLC).

RESULTS: Maximum extraction of polyphenols (407.47 mg GAE g1), total tannin and its antioxidant activity with minimumarecoline (1.73mg g1 of sample) was achieved by using 80% acetone at pH 4 for 90minwith 10%w/v substrate under shakingconditions.

CONCLUSION: Solvent extraction under optimized parameters gave maximum polyphenols with minimum extraction ofarecoline, and highest ratio of polyphenols to arecoline. HPLC and liquid chromatographymass spectrometry resultsconfirmed the presence of catechin and epicatechin in the extract, which suggests its potential as a source of bioactives.c 2013 Society of Chemical Industry

Keywords: arecanut; polyphenols; arecoline; antioxidant activity; solvent extraction

INTRODUCTIONArecanut or betel nut (Arecacatechu L.) is an important commercialcrop grown in the humid tropics of India. It is traditionally usedfor masticatory purposes and also in ayurvedic and veterinarypreparations. Commercially, it is mainly used in gutka and panmasala industries and its consumption is intimately associatedwith religious, social andcultural ceremonies. Arecanut is generallyproduced in two forms: white supari and red supari. It is asource of sustainable income for small landowners. Globally, Indiaaccounts for 58% and 53% of the total area and production ofarecanut, respectively. Chemical constituents present in arecanutare carbohydrates, lipids, proteins, crude fibres, polyphenols(flavonols and tannins) and minerals.1 It also contains 0.21.7%alkaloids.2 Physiologically, the most important constituents ofarecanut are alkaloids and polyphenols. Dande and Manchala3

studied antioxidant activity as well as phenolic content of nuts,oilseeds, milk and milk product and found arecanut to be a richsource of phenolics.

Polyphenols are generally divided into hydrolysable tannin andcondensed tannins. Arecanut fruit is known to contain condensedtannins, hydrolysable tannin, simple phenolics and structurallyrelated monomer and oligomeric flavan-3-ols.4 The content ofphenolics and condensed tannins is reported to increase whilethat of arecoline is reported to decrease on maturity.5 Arecanutcontains mainly flavonoids and polymerized leucocyanidins,

besides minor amounts of (+)-catechin, leucopelargonidin andleucocyanidin.Theyareknowntoprotectoxidationofhigh-densitylipids (HDLs). Its constituents show anthelmentic, antifungal,antibacterial, anti-inflammatory, antioxidant, insecticidal andlavicidal activity.6 Arecanut has been enlisted for its effects againstleucoderma, cough,fits, anaemia,obesityandcertain skindiseases.Areca tannin has been suggested to regulate blood pressure byinhibiting the pressor response to both angiotensin I and II.7

Polyphenols have been documented to show anti-inflammatory,anti-allergic, antiviral and anticancer activities.8

Arecanut contains four alkaloids belonging to pyridine group,namely arecoline, arecaidine, guvacine and guvacoline, of whichthemost importantandpotent is arecoline.2 It actsasanagonist forthe muscarinic receptor, stimulates parasympathetic action, andalso shows positive cardiovascular and ocular effects.9 Immaturearecanutsareknowntocontainall fouralkaloids,whilematureonescontain only arecoline.5 The widespread consumption and use ofarecanut or its products raises concerns about the nutritional and

Correspondence to: Rekha S Singhal, Food Engineering and TechnologyDepartment, Institute of Chemical Technology, Matunga, Mumbai 400019,India. E-mail: rs.singhal@ictmumbai.edu.in

Food Engineering and Technology Department, Institute of ChemicalTechnology, Matunga, Mumbai 400019, India

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Table 1. Effect of solvent systems on extraction of arecanut constituents

Solvents

Total phenoliccontent (TPC)(mg GAE g1)

Total tannin content(TTC)

(mg CE g1)Arecoline(mg g1)

TPC:arecoline

ABTS(mmol L1 TEAC)

FRAP(mmol L1 TEAC)

Hydroxylscavenging activity (%)

Water 112.66 0.99a 4.28 0.13a 6.38 0.02a 17.66 81.35 1.03a 763.06 1.3a 20.40 1.06aMethanol 198.53 0.43b 39.43 0.28b 12.79 0.00b 15.52 409.23 2.17b 928.89 1.4b 20.71 1.79bEthanol 170.67 0.07c 39.21 0.34b 9.79 0.05c 17.43 405.18 2.76c 916.11 2.1c 14.01 0.75cAcetone 218.29 0.68d 53.34 0.78c 2.30 0.01d 94.90 422.73 0.24d 947.50 2.2d 28.18 0.82dChloroform 12.32 0.18e 0.001 0.08d NDe 0.00 NDe NDe NDeEthyl acetate 72.02 1.69f 29.50 0.54e NDf 0.00 40.09 0.05f 17.78 1.5f NDfHexane 36.55 0.52g 0.01 0.00d NDg 0.00 NDg NDg NDgn-Butanol 41.07 0.18h 0.01 0.01d NDh 0.00 NDh NDh NDhData are expressed as mean standard deviation of triplicate experiments (n= 3). Mean values in the same column followed by the same letter donot differ significantly (P< 0.05).ND, not detected.

health consequences, in particular oral cancer. This is mainly dueto arecoline, which is metabolized to nitroso compounds, whichare known to be cytotoxic and genotoxic to human buccal epithe-lial cells10 and could also produce pancreatic, lung, nasal and livertumours in rats.11 TheWorldHealthOrganizationand InternationalAgency for Research on Cancer classified arecanut as a Group 1human carcinogen. In contrast, arecoline also has potential appli-cation in Alzheimers disease due to its potent muscarnic action. Itis also used to treat severe depression and schizophrenia.12

Commercial useof arecanutwithout its toxic effects necessitatesthedevelopmentof efficient extractionand separationofpolyphe-nols from alkaloids. Although some investigators have reportedon the extraction of polyphenols from areacnut,5,13 they have nei-ther looked into the alkaloids which are co-extracted along withpolyphenolsnor theantioxidantactivityof theextractedphenolics.Thus the present study aimed to develop a protocol for maximumextraction of beneficial polyphenols from mature arecanut withminimum extraction of arecoline. This will open newer vistas tothose industries that manufacture products from arecanut.

MATERIALS ANDMETHODSMaterialsMangalore variety of arecanut was used in the present study andpurchased from an authorized market in Mumbai, India. It wasground in a stainless steel grinder (New India Trading Company,Mumbai) to a powder passing through 40-mesh size and stored inairtight containers until further use. Catechin 99%, phenanthroline

and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)were purchased from Sigma-Aldrich Chemicals, Mumbai, India.Arecoline hydrobromide (alkaloid) was procured from FisherScientific Chemicals, Mumbai. 2,4,6-Tripyridyl-s-triazine (TPTZ),FolinCiocalteu reagent and all solvents (analytical grade) wereprocured from SD Fine Chemicals, Mumbai, India.

Optimization of the extraction procedureEffect of solventGround, moisture-free and defatted arecanut powder (10% w/v)was extracted with water, methanol, ethanol, acetone, ethylacetate, chloroform,hexaneandn-butanol at 180 rpmonanorbitalshaker for 1 h. The extractwas centrifuged at 7000 g for 20min at25 Candfiltered throughWhatmanNo. 1filter paper. Supernatantwas collected, evaporated in a RotaVac (Buchi rotavapor R-124,Flawil, Switzerland) at 58 C to a final volume of 2 mL and storedin the dark at 4 C for further analysis. The extracts were analysedfor total phenolic content (TPC), condensed tannin content (TTC),arecoline, antioxidant activity and TPC:arecoline ratio.

Since acetone gave the best extraction of polyphenols withminimumarecolinecontent, furthereffectof100%to20%aqueousacetone on the same was evaluated. All experiments were carriedout in triplicate.

Effect of extracting conditionsThe effect of pH on extraction of TPC, TTC, arecoline, antioxidantactivity and TPC:arecoline ratio was studied by varying the pH ofextracting solvent from 2 to 10. The pH of the extract was adjusted

Table 2. Effect of aqueous acetone on extraction of arecanut constituents

Aq. acetone

(%, vol.)

Total phenolic

content (TPC)

(mg GAE g1)Total tannin content

(TTC) (mg CE g1)Arecoline

(mg g1)TPC:

arecoline

ABTS

(mmol L1 TEAC)FRAP

(mol L1 TEAC)Hydroxyl scavenging

activity (%)

100 218.30 0.90a 53.60 0.78a 2.47 0.01a 88.38 417.24 0.85a 946.56 2.01a 28.18 0.81a80 297.30 0.96b 68.53 0.61b 3.43 0.02b 86.67 429.80 0.43b 997.28 1.12b 53.34 0.17b60 208.41 0.72c 41.28 0.26c 4.50 0.01c 46.31 404.43 0.43c 963.89 1.17c 38.63 0.17c40 196.82 0.59d 27.73 0.58d 4.62 0.01c 42.60 382.51 0.85d 850.28 1.30d 27.77 0.29a20 171.74 1.07e 20.48 0.40d 5.64 0.02d 30.27 349.26 1.01e 601.67 1.01e 24.77 0.68dData are expressed as mean standard deviation of triplicate experiments (n= 3). Mean values in the same column followed by the same letter donot differ significantly (P< 0.05).

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Table 3. Effect of pH on extraction of arecanut constituents

pH value

Total phenolic

content (TPC)

(mg GAE g1)Total tannin content

(TTC) (mg CE g1)Arecoline

(mg g1)TPC:

arecoline

ABTS

(mmol L1 TEAC)FRAP

(mmol L1 TEAC)Hydroxyl scavenging

activity (%)

2 181.66 1.37a 65.01 0.08a 0.55 0.01a 330.29 440.26 1.29a 373.61 2.40a 42.23 0.58a4 315.07 1.38b 81.15 0.41b 1.24 0.02b 254.09 458.82 0.1b 1010.14 0.26b 49.78 0.44b6 320.63 1.20c 70.01 0.47c 4.19 0.01c 76.52 459.83 0.43b 1011.89 2.01b 52.18 0.07c8 165.07 1.31d 44.12 0.20d 5.47 0.01d 30.17 434.07 2.61c 920.28 2.12c 38.98 0.77d10 113.49 1.37e 34.21 0.31e 5.60 0.14e 20.26 424.91 1.13d 872.22 1.3d 36.04 0.84eData are expressed as mean standard deviation of triplicate experiments (n= 3). Mean values in the same column followed by the same letter donot differ significantly (P< 0.05).

(A)

(B)

Figure1. (A) Effect of pHon correlationbetweenpolyphenolic content andantioxidantactivityof arecanutextract. (B) Effectof substrateconcentrationon correlation between polyphenolic content and antioxidant activity ofarecanut extract.

using 1mol L1 HCl or 1mol L1 NaOH. The effect of concentrationof arecanut powder on extraction was investigated by varying thesample concentration from6%to14%w/vandevaluatedasabove.Inorder to investigatetheeffectofextractiontime, theexperimentswere carried out on orbital shaker at 180 rpm, and samples drawnand processed at regular interval and analysed as above.

Analytical methodsDetermination of total phenolic contentTPCwas determined using the FolinCiocalteumethod describedbySingletonandRossi14 withsomemodifications.0.2mLofdilutedextracts were mixed with 1 mL of 1:10 diluted FolinCiocalteureagent and reacted for 5 min. 0.8 mL of 7.5% sodium carbonatewas added to the mixture, and incubated for 30 min in the darkat 27 2 C. Absorbance was measured at 765 nm on a Hitachi

spectrophotometer (Model U-2001). A standard graph preparedusing gallic acid in the range 20250 g mL1 gave a regressionequationY = 0.0021X (R2 = 0.9917) thatcorrelated theabsorbanceat 765 nm (Y) and gallic acid (X). TPC content was expressed asmilligrams of gallic acid equivalents (GAE) per gram of sample.

Determination of condensed tannin contentThe vanillinHCl assay, whichmeasures the amount of condensedtannins present in the sample, was done according to Nakamuraet al. with somemodifications.15 Briefly, 1mL of samplewasmixedwith 5 mL vanillin reagent (8% HCl in methanol and 4% vanillinin methanol, 1:1 v/v) and incubated at 27 2 C for 20 min.Absorbance was measured at 500 nm on a UV-visible spectropho-tometer (Helios, Thermo Electron, Dreieich, Germany). A standardgraph was prepared using catechin in the concentration range50250 g to give a regression equation Y = 0.0015X (R2 = 0.995)that correlated the absorbance at 500 nm (Y) and catechin (X).The results were expressed as milligrams of catechin equivalents(CE) per gram of sample.

Determination of ferrous reducing antioxidant power assay (FRAP)FRAP assay was conducted according to the method of Benzieand Strain,16 with some modification. Briefly, a 100 L aliquot ofarecanut extractwasmixedwith 3mLFRAP reagent and incubatedat 27 2C for 8min, after which the absorbancewasmeasured at593 nm against a blank. FRAP reagent should be pre-warmed at 37Cprior touseandshouldbefreshlypreparedbymixing0.2mol L1

acetate buffer (pH 3.6), 10 mmol L1 TPTZ in 40mmol L1 HCl and20mmolL1 FeCl3 ina10:1:1 ratio.AstandardcurvepreparedusingTrolox in the range 50500 mol L1 gave a regression equationY = 0.001X (R2 = 0.997), which correlated the absorbance at 593nm (Y) and Trolox (X). The final results were expressed as Troloxequivalent antioxidant capacity (TEAC) per gram of sample.

Determination of ABTS radical scavenging activityAntioxidant power of arecanut extract was assessed according tothe ABTS assay described by Abraham and Mathew.17 The ABTSradical cations (ABTS.+)wasproducedbyreacting7mmolL1 stocksolution of ABTS with 2.45 m mol L1 potassium persulfate (finalconcentration) and allowed to stand in the dark for at least 16 h at27 2 C before use. The ABTS solution was diluted using ethanolto an absorbance of 0.7 0.06 at 734 nm (Hitachi UV-visiblespectrophotometer, U-2001). 0.1 mL arecanut extract was mixedwith 3.9 mL ABTS reagent and absorbance was measured exactlyafter 6 min. A standard graph prepared using Trolox in the range

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Table 4. Effect of substrate concentration on extraction of arecanut constituents

Arecanutpowderconcn (%, wt)

Totalphenolic content

(TPC) (mg GAE g1)Total tannin content(TTC) (mg CE g1)

Arecoline(mg g1)

TPC:arecoline

ABTS(mmol L1 TEAC)

FRAP(mmol L1 TEAC)

Hydroxylscavengingactivity (%)

6 216.93 1.31a 66.26 0.83a 1.14 0.01a 190.29 224.76 2.06a 382.78 1.30a 26.95 0.11a8 255.80 0.01b 71.20 0.74b 1.24 0.01b 206.29 263.15 1.60b 590.01 1.02b 31.01 0.80b10 320.28 1.01c 78.53 1.07c 1.34 0.02c 239.01 458.62 1.20c 1011.44 1.11c 52.77 0.24c12 381.13 1.67d 101.53 0.20d 2.09 0.02d 182.36 478.86 1.03d 1042.78 1.21d 60.12 0.46d14 396.22 1.25e 104.71 0.48d 2.58 0.01e 153.57 502.06 1.20e 1061.67 1.14e 62.32 0.01eData are expressed as mean standard deviation of triplicate experiments (n= 3). Mean values in the same column followed by the same letter donot differ significantly (P< 0.05).

Table 5. Effect of extraction time on arecanut constituents

ExtractionTime (min)

Total phenoliccontent (TPC)(mg GAE g1)

Total tannincontent (TTC)(mg CE g1)

Arecoline(mg g1)

TPC:arecoline

ABTS(mmol L1 TEAC)

FRAP(mmol L1 TEAC)

Hydroxylscavenging activity (%)

30 212.30 2.38a 55.55 1.66a 0.86 0.01a 246.86 216.57 1.13a 689.17 1.40a 14.90 0.13a60 399.47 2.70bc 96.57 0.77b 1.24 0.02b 322.15 480.73 1.08b 1038.06 1.51b 55.42 1.10b90 407.47 1.31b 101.82 2.82c 1.73 0.03c 235.53 504.71 1.20c 1043.33 0.83c 61.44 1.09c120 391.53 1.37bc 102.62 1.92c 2.46 0.01d 159.16 502.51 1.23c 1030.83 1.23d 52.88 0.46d150 381.61 1.37bc 99.95 0.50bc 2.66 0.01e 143.46 476.27 1.12d 1003.28 1.21e 41.46 0.30eData are expressed as mean standard deviation of triplicate experiments (n= 3). Mean values in the same column followed by the same letter donot differ significantly (P< 0.05).

050molL1 gavea regressionequationY = 1.069X (R2 = 0.990),which correlated the absorbance at 734 nm (Y) and Trolox (X).Antioxidant power was calculated as percentage inhibition:

%inhibition = Ablank AextractAblank

100

where Ablank and Aextract was the absorbance of the blank(ethanol) and the extract after 6 min. The final results wereexpressed as TEAC per gram of sample.

Determination of hydroxyl radical scavenging activityThis assay was carried out according to De Avellar et al.18 withsome modifications. 3.75 mmol L1 1,10-phenanthroline and7.5 mmol L1 ferrous sulfate were prepared in 0.1 mol L1

phosphate buffer (pH 7.4). To the test tubes containing 0.4 mL1,10-phenanthroline and 0.2 mL ferrous sulfate, about 0.8 mLof 0.05% hydrogen peroxide was added. To the mixture, 0.1mL of the arecanut extract was added and incubated at 37 Cfor 60 min. The blank and control sample were prepared withacetone. The absorbance of the mixture was measured at 536nm using a Helios () UV-visible spectrophotometer (ThermoElectron, Osterode, Germany). A standard graph prepared usingbutylated hydroxytoluene (BHT) in the range 0.11 g gave aregression equation Y = 119.14X (R2 = 0.9972), which correlatedthe absorbance at 536 nm (Y) and BHT (X). The percent hydroxylradicals scavenged was calculated from the following equation:

% hydroxyl radical scavenged = [(AS A1) / (A0 A1)] 100

where, AS, A1 and A0 are the absorbance of the sample, controlsolution containing 1,10-phenanthroline, FeSO4 and H2O2 andblank solution containing 1,10-phenanthroline and FeSO4.

Determination of arecoline content by HPLCArecoline contentof theextractwasdeterminedbyHPLCusing themethod described by Aromdee et al.,19 with some modification.The Jasco HPLC system fitted with UV-visible detector (UV-1575)at 216 nm was used at an isocratic flow of 0.8 mL min1 at45 C. The detection of analytes was accomplished with a C18column (Spherisorb ODS2, 5 m, 4.6 250 mm analytical column,Waters,Milford,MA,USA). Themobilephasecompositionusedwaspotassium dihydrogen phosphate (10 g L1), phosphoric acid (3.5mL L1), triethylamine (8mL L1) and acetonitrile (12mL L1), andwas filtered through 0.22 before use. The pHof themobile phasewas adjusted to 4.5 with either triethylamine or phosphoric acid.A standard graph was prepared using arecoline hydrobromide(99%) in the range 1090 g. The regression equation correlatingthe area under the peak (Y) and arecoline (X) was Y = 60326X(R2 = 0.998).

Identification of polyphenols by HPLCThe analysis of polyphenols was performed using HPLC (Flexar600, PerkinElmer, Waltham, MA, USA) with a Hypersil C18 column(Brownlee Analytical C18, 4.6 m, 4.6 150mm analytical column)of length 250 mm using a UV-visible detector at 280 nm. The col-umnwas equilibratedwith acetonitrile (B)0.1% orthophosphoricacid (A). The column was mounted on a PerkinElmer Flexar 200pumpHPLCchromatographequippedwith20L loop injector.Thegradient analysis was carried out for 33 min at 25 C. The multilin-ear gradient beganwith 100%Aup to3min, 90%A+ 10%B from3

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Epicatechin

Catechin

Figure 2. HPLC chromatogram of arecanut crude extract.

to 6.5min, 75%A+ 25%B from 6.5 to 14min, 45%A= 55%B from14 to 25 min, and finally with 10% A+ 90% B from 25 to 33 min.

Identification of arecanut constituents using LCMSA Varian LCMS-500L system equipped with ion trap mass analyserhaving a mass range from 20 to 2000 amu, photodiode arraydetector (PDA), autoinjector, and LC binary pump coupled onlinewith anMS-500mass spectrometer (Varian Inc., PaloAlto, CA,USA).Electrospray ionization (ESI) was operated in the negative mode.Ion spray voltage was set at 4000 V and orifice voltage 60 V.HPLC separationwas carried out on a narrow-bore reversed-phasecolumn (C18). The column was connected with a mass interfacevia fused-silica capillary tubing (length 100 cm, 100 m i.d.). Thesample was injected with a rotary valve fitted with 20 L sampleloop. Elution was achieved using the mobile phase acetonitrile(A):water (B). Gradient analysis was carried out for 15 min. Thegradient began with 100% B up to 1 min, from 1 to 5 min 50%A+ 50% B, from 5 to 10 min 75% A+ 25% B and finally with 100%A+ 0% B from 10 to 15 min.

Statistical analysisAll data were expressed as means standard error of triplicatemeasurements and analysed by SPSS for Windows (version16.0, SPSS Inc., Chicago. IL. USA). One-way analysis of variance(ANOVA): post hoc multiple comparisons were carried out to testany significant differences between solvents used, solvent:waterratio, pH, substrate concentration and extraction time. Statisticalcomparisons between variables (e.g. TPC, TTC, arecoline andantioxidant activity) were performed using the least squaredifferential method (LSD). Differences were considered significantat P< 0.05.

RESULTS ANDDISCUSSIONEffect of solventVarious polar and non-polar solvents such as water, methanol,ethanol, n-butanol, acetone, chloroform, ethyl acetate and hexanewere used for the extraction of TPC, TTC and arecoline from driedarecanut powder. Among these, acetone resulted in maximumextraction of polyphenols (218.29 mg GAE g1 of sample) andtannin (53.34 mg CE g1 of sample). Interestingly, acetone alsogave minimum extraction of arecoline (2.30 mg g1 of sample),whereas methanol resulted in maximum extraction followed byethanol (Table 1). These results indicate arecoline to be solublein water and alcohol and are in agreement with those of

Chiang et al.20 Naczk and Shahidi21 reported the extraction ofphenolic compounds to depend on the solubility and polarity ofsolvents. Furthermore, acetone also showed a maximum ratio ofTPC:arecoline, which again signified the extraction of maximumpolyphenols with minimum arecoline.

Besides TPC, antioxidant assays suchasABTS, FRAPandhydroxylradical scavenging activity were carried out to determine theantioxidant potency of extracts obtained. Maximum ABTS (422.73mmol L1 TEAC g1 of sample), FRAP (947.50 mmol L1 TEACg1 of sample) and hydroxyl radical scavenging activity (28.18%)was also seen with the acetone extract. This was followed bymethanol, ethanol and water, which were parallel to the phenoliccontent (Table 1). Pan et al.22 reported a correlation betweenphenolic compounds and antioxidant activities. In our work too,a correlation between the content of phenolic compound andantioxidant activity was observed. Since the solvent influencesthe extraction of polyphenols, it also had a similar effect on theantioxidant profile.

Further extraction of phenolic compounds was carried outusing acetone with varying proportions of water. It is evidentfrom Table 2 that 80% acetone gave the highest TPC (297.30mg GAE g1 of sample), and TTC (68.53 mg CE g1 of sample)with a concomitant increase in arecoline (3.43 mg g1 of sample),which can be accounted for by the presence of water in thesolvent mixture. The phenolic content decreased at a watercontent above 20%, which can be attributed to lower solubilityof phenolics in water. Alcohol water mixtures are known to bemore efficient than the corresponding mono-component solventsystem in extracting phenolic constituents.23 The maximum ratioof TPC:arecoline was observed in 80% acetone. It is observed fromTable 2 that ABTS, FRAP and hydroxyl radical scavenging activitydecreased with an increased proportion of water content in theacetone:water blend and maximum activity was observed in thesolvent blend containing 80% acetone. This may be due to achange in solvent polarity, which helps to dissolve the selectedgroup of antioxidant compounds and influences the antioxidantactivity.24 The antioxidant activity showed significant correlationwith TPC. The maximum ABTS (429.80 TEAC g1 of sample), FRAP(997.28 mmol L1 TEAC g1 of sample), and hydroxyl radicalscavenging activity (53.34%) and phenolic content was observedin solvent containing 80% acetone. Yilmaz and Toledo25 found thephenolic content of ethanolic extract from grape seed to increasewith increase in water from 0 to 30% in themixture and decreasedwith a further increase in water. Similar results were reportedby Cacace and Mazza26 on blackcurrant using aqueous ethanol

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100 150 200 250 300 350 400 m/zR.Match: 578, F.Match: 426

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F F F

105.15051 133.23678

149.111190

152.22095

156.237037

172.27659

203.214992

207.26882 225.3

5309 245.32697

291.22824

Spectrum 1A1.836 min, Scan: 125, 100:400, Ion: 60198 us, RIC: 400566BP: 156.2 (37037=100%), -crude extract 5-25-2012 12-30-05 pm.xms

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F

107.13200

123.114346

127.16001

133.21399

139.120603

147.26821

152.11278

173.13776 193.3

2262

207.311275

215.33226 233.3

1594

247.36486

259.22371

287.34194

Spectrum 1B7.301 min, Scan: 495, 100:400, Ion: 115371 us, RIC: 310540BP: 139.1 (20603=100%), -crude extract 5-25-2012 12-30-05 pm.xms

N OOCH3H3C

(A)

Figure 3a. HPLC-ESI-MS of the sample extracted from arecanut: ion chromatogram extracted atm/z value corresponding to the M+H ion of arecoline.

and found 70% ethanol to be the best for extraction of phenoliccompound.Hence80%acetonewasused for further studies.Wangand Hwang27 also used 80% acetone for extraction of phenoliccompound from betel quid using different extraction protocols,although they did not estimate the content of arecoline or theantioxidant activity of betel nut. Han et al.28 extracted TPC frombetel nut with 70% ethanol using ultrasound-assisted extraction.

Effect of extraction conditionsArecanut powder was extracted with 80% aqueous acetone atdifferent pH values and analysed for TPC, TTC, arecoline andantioxidant activity. The extraction of TPC and TTC increased withan increase in pH from 2 to 6 and decreased sharply above pH 6(Table 3), while that of arecoline also increased with an increasein pH from 2 to 10. A pH of 4 showed good extraction of TPC(315.07mg GAE g1 sample) and of TTC (81.15mg CE g1 sample)

with minimum arecoline (1.24 mg g1). The variation observed inphenolic content with change in pH may be accounted for by theselective solubility characteristics of some polyphenols at thesepH values.29 The ratio of TPC:arecoline also varied with pH andwas found to bemaximal at pH 2. Since the total phenolic contentwas lower at pH 2, TPC:arecoline ratio at pH 4 was considered tobe the optimumwhich resulted in higher phenolic content with alower amount of arecoline.

In addition to phenolic content, extract was analysed forantioxidant potency. The antioxidant capacity of arecanut extractwas exhibited by high ABTS (458.82 mmol L1 TEAC g1 ofsample), FRAP (1010.14 mmol L1 TEAC g1 of sample) andhydroxyl radical scavenging activity (49.78%) at pH 4 and canpositively be correlated with phenolic content (Table 3). A changein colour of the extract was also observed with an increase in pH.This could be due to precipitation of the coloured pigment

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kCounts -CRUDE EXTRACT 5-25-2012 12-30-05 PM.XMS 100:400 (-) Filtered100:400 (-)

1A 1B

100 150 200 250 300 350 400 m/zR.Match: 116, F.Match: 6

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1

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124.92862

136.9531

160.91702

176.9558

203.0600

221.1608

245.11441

255.2339 271.1214

287.1605

289.03874

Spectrum 1A6.997 min, Scan: 472, 100:400 (-), Ion: 141755 us, RIC: 23754BP: 289.0 (3874=100%), -crude extract 5-25-2012 12-30-05 pm.xms

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207.112590

208.11461 227.1

940255.1862

325.11770

Spectrum 1B11.937 min, Scan: 816, 100:400 (-), Ion: 206675 us, RIC: 24301BP: 207.1 (12590=100%), -crude extract 5-25-2012 12-30-05 pm.xms

Catechin: 289 (M+H)

HO

HO

O

OHOH

OH

(B)

Figure 3b. HPLC-ESI-MS of the sample extracted from arecanut: ion chromatogram extracted atm/z value corresponding to the M+H ion of catechin.

or alteration in chromophoric characteristics under alkalineconditions.30

In these sets of experiments too, the extract of arecanutshowed positive correlation between antioxidant activity (ABTS,FRAP and hydroxyl radical scavenging activity) and polyphenolconcentration (Fig. 1A), which is in agreement with previousreports for tea polyphenols and antioxidant activity.31 Thecorresponding correlation coefficients were 0.984 for ABTS(y = 0.157x + 408.6), 0.99 for FRAP (y = 9.415x 692.45) and 0.978for hydroxyl scavenging activity (y = 0.073x + 27.78).

The effect of sample concentration (614%) on extraction ofTPC, TTC and arecoline showed 14% to extract maximum TPC(396.22mg GAE g1 of sample), TTC (104.71mg CE g1 of sample)and arecoline content (2.58 0.01 mg g1 of sample) (Table 4).A significant difference was observed in TPC at 10%, 12% and14% w/v of sample but, with an increase in the concentration ofarecanut, extraction of arecoline also increased. The TPC:arecoline

ratio also increased with an increase in concentration andmaximum TPC:arecoline ratio (239.01) was observed with 10%w/v. Hence 10% concentration was optimized for further study.

Furthermore, ABTS (458.62 mmol L1 g1 of sample), FRAP(1011.44 mmol L1 TEAC g1 of sample) and percent hydroxylradical scavenging activity (52.77%) at 10%w/vwas observed. Thetrend of antioxidant activity was similar to that of TPC. This maybe due to the ability of antioxidants to scavenge certain radicalswith higher concentration.32 To evaluate the experimental results,attemptsweremade to correlate the content of polyphenols in theextracts with their antioxidant activity (Fig. 1B). TEAC was foundto be concentration-dependent and agrees with a previous workon linear correlation between TPC and antioxidant activity.33 TheABTS, FRAP and hydroxyl scavenging activity also showed goodcorrelation with TPC. The correlation coefficients were 0.916 forFRAP (y = 3.089 378.54), 0.922 for ABTS (y = 1.618x 122.8) and0.967forpercenthydroxylscavengingactivity (y = 0.210x 19.45).

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www.soci.org Y V Chavan, R S Singhal

75 100 125 150 175 200 m/z

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171.911

178.826

199.06

Spectrum 1A0.233 min, Scan: 17,199.0>75:209 (-) [1.20V], Ion: 250000 us, RIC: 43BP: 178.8 (26=100%), sample-199-msms.xms

(a) Arecoline N oxide: 171

(b) Syringic acid orArecaidinylglycine199 (M+H)

N+

-OCH3

O OH

(C)

Figure 3c. HPLC-ESI-MS of the sample extracted from arecanut: ion chromatogram extracted at m/z value corresponding to the M+H ion of (a)arecoline-N-oxide and (b) arecaidinylglycine or syringic acid.

In order to investigate the effect of extraction time, the samplewas kept on an orbital shaker at 180 rpm, pH 4 and 10% sampleconcentration. Samples were drawn at regular intervals of 30min and the extract was analysed for TPC, TTC, arecoline andantioxidant activity. The extraction of TPC and TTC from arecanutincreasedwith an increase in extraction time up to 90min andwassignificantly altered after 120 and 150 min of extraction (Table 5).Optimum extraction of TPC and TTC with an adequate amountof arecoline was observed at 90 min. The amount of arecolinealso increased with an increase in extraction time, and maximumTPC:arecoline ratiowas found at 60min. Lapornik etal.34 found theyield of grape polyphenols to increase with time, while in the caseof alcohol extracts it strongly increased with extraction time up toa certain limit and then decreased. A TPC:arecoline ratio (235.53)was observed after 90 min of extraction.

Furthermore, antioxidant potency of the extract was foundto be optimal at 90 min. Activity of ABTS (504.71 mmol L1

per gram of sample), reducing power of FRAP (1043.33 mmolL1 per gram of sample) and scavenging activity of hydroxyl

radical (61.44%) increased up to 90 min and decreased thereafter(Table 5). Hence 90 min of extraction time was optimized forfurther studies. This may be due to the degradation or oxidationof phenolics on prolonged extraction. Perva-Uzunalic et al.35

reported on extraction of catechin with water from green tea andobtainedmaximumextraction efficiency after 20min. Yang et al.36

studied various extraction parameters on Phyllanthus emblica L.for extraction of polyphenols and among all parameters foundsolvent concentration (75% ethanol) and extraction time (45 min)to have a significant effect on polyphenol extraction.

Identification of arecanut bioconstituents by HPLC and LC-MSPolyphenols like catechin and epicatechin were tentativelyidentified based on HPLC and comparison of their electronicabsorption spectra with authentic standards. For identificationof polyphenolic compounds, HPLC was used with a UV detectorat 280 nm. Two major peaks eluting at 4.5 and 7.11 min ina total run time of 33 min were seen in the chromatogramof crude extract of arecanut (Fig. 2). Individual standards of

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Separation of polyphenols and arecoline from areca nut www.soci.org

catechin and epicatechin injected to check the presence ofeither or both of these compounds confirmed the presenceof catechin (4.5 min) and epicatechin (7.14 min) in arecanutextract. For further confirmation, LC-MS was performed with theextract.

Mass spectrometry is a highly sensitive technique requiring verysmall amounts of material (typically 1 g) for analysis. The processof elucidating the mass and the structure of a substance can beexpedited by using a high-performancemass spectrometer. UsingESImassspectrometry,whichproducedprotonatedmolecular ionswith minimal or no fragmentation, pseudo-molecular ions of eacheluting species can be readily produced. ESI mass spectrometryis superior to others in this respect since structurally significantinformation can be elicited by collisional activation of the elutingspecies in the vacuum interface of the ESI source.

The presence of unchanged arecoline in crude extract wasdetermined from the occurrence of an ion of [M+H] + = 156m/z which gave a match for C8H13NO2 (Fig. 3a). The presence ofcatechin and epicatechin was also identified by the occurrenceof [M+H]+ = 289.0 m/z and which gave a match for C9H14N2O3(Fig. 3b). Giri etal.37 reported thepresenceof arecolineNoxide andarecaidinylglycine from the presence of an ion of [M+H]+ = 172m/z (C8H14NO3) and [M+H] + = 199.1 m/z, respectively, duringtheir study on ametabolic approach to arecanut alkaloids, namelyarecoline and arecaidine, in mouse urine. These compoundsare probably present in arecanut extract as metabolic productsdue to indigenous plant enzymes. The peak corresponding to[M+H]+ = 199.1m/zmayalsobeattributed to syringicacid,whichhas been reported by Zhang et al.38 at the same [M+H]+ = 199.1m/z. However, the major peak observed in Fig. 3c could not beidentified. The presence of acid in the mobile phase is essentialfor complete separation and elution of these analytes.37 Besidesthese, 13 other compounds which could not be identified werealso obtained.

In conclusion, maximum extraction of polyphenols with mini-mum extraction of arecoline was obtained using 80% acetone ofpH 4 with 10% arecanut concentration for 90 min. The extract soobtained gave a TPC of 407.47 mg GAE g1 of sample, a TTC of101.82 mg CE g1 of sample, and arecoline at 1.73 mg g1 of sam-ple. The antioxidant activities of the extracts, as seen from ABTS,FRAP and hydroxyl scavenging assays, correlated with the totalphenolics. Separation and identification of individual componentsin arecanut was confirmed using HPLC and LC-MS. The presenceof catechin, epicatechin and arecoline in arecanut extractwas con-firmed fromHPLC and LC-MS chromatograms. These results couldbe useful in obtaining arecoline-free extraction from arecanut andusing the otherwise toxic bioresource for beneficial purposes. Fur-ther work on minimizing the extraction of arecoline is in progress.

ACKNOWLEDGEMENTWeare thankful to theUniversity Grants Commission, Governmentof India, New Delhi, for providing a fellowship to the first authorand financial support for the project.

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