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Received: 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 from
areca nut (Areca catechu L.) by solvent
extraction, its antioxidant activity, and
identication of polyphenols
Yogita V Chavan and Rekha S Singhal
Abstract
BACKGROUND: Arecanut (ArecacatechuL.) or betel nut, acommercial cashcrop, is arichsourceof polyphenols but alsocontains
toxic alkaloids, mainly arecoline. Separation of these bioactive polyphenols from toxic constituents could propel the safe and
benecial use of betel nut; also it will help arecanut processing industries to produce arecoline-free products. With the aim to
develop an effective method for maximum extraction of polyphenols with minimum arecoline, several factors such as nature
of the solvent, pH (210), substrate concentration (614 %) and extraction time (30150 min) under shaking conditions were
evaluated. Qualitative analysis was done using spectrophotometry and high-performance liquid chromatography (HPLC).
RESULTS: Maximum extraction of polyphenols (407.47 mg GAE g
1
), total tannin and its antioxidant activity with minimum
arecoline (1.73 mg g
1
of sample) was achieved by using 80%acetone at pH4 for 90 min with 10%w/v substrate under shaking
conditions.
CONCLUSION: Solvent extraction under optimized parameters gave maximum polyphenols with minimum extraction of
arecoline, and highest ratio of polyphenols to arecoline. HPLC and liquid chromatographymass spectrometry results
conrmed 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
INTRODUCTION
Arecanut or betel nut (ArecacatechuL.) is animportant commercial
crop grown in the humid tropics of India. It is traditionally used
for masticatory purposes and also in ayurvedic and veterinary
preparations. Commercially, it is mainly used in gutka and pan
masala industries and its consumption is intimately associated
withreligious, social andcultural ceremonies. Arecanut is generally
produced in two forms: white supari and red supari. It is a
source of sustainable income for small landowners. Globally, India
accounts for 58% and 53% of the total area and production of
arecanut, respectively. Chemical constituents present in arecanut
are carbohydrates, lipids, proteins, crude bres, polyphenols
(avonols and tannins) and minerals.
1
It also contains 0.21.7%
alkaloids.
2
Physiologically, the most important constituents of
arecanut are alkaloids and polyphenols. Dande and Manchala
3
studied antioxidant activity as well as phenolic content of nuts,
oilseeds, milk and milk product and found arecanut to be a rich
source of phenolics.
Polyphenols are generally divided into hydrolysable tannin and
condensed tannins. Arecanut fruit is known to contain condensed
tannins, hydrolysable tannin, simple phenolics and structurally
related monomer and oligomeric avan-3-ols.
4
The content of
phenolics and condensed tannins is reported to increase while
that of arecoline is reported to decrease on maturity.
5
Arecanut
contains mainly avonoids and polymerized leucocyanidins,
besides minor amounts of (+)-catechin, leucopelargonidin and
leucocyanidin. Theyareknowntoprotect oxidationof high-density
lipids (HDLs). Its constituents show anthelmentic, antifungal,
antibacterial, anti-inammatory, antioxidant, insecticidal and
lavicidal activity.
6
Arecanut has been enlisted for its effects against
leucoderma, cough, ts, anaemia, obesityandcertainskindiseases.
Areca tannin has been suggested to regulate blood pressure by
inhibiting the pressor response to both angiotensin I and II.
7
Polyphenols have been documented to show anti-inammatory,
anti-allergic, antiviral and anticancer activities.
8
Arecanut contains four alkaloids belonging to pyridine group,
namely arecoline, arecaidine, guvacine and guvacoline, of which
themost important andpotent is arecoline.
2
It acts as anagonist for
the muscarinic receptor, stimulates parasympathetic action, and
also shows positive cardiovascular and ocular effects.
9
Immature
arecanutsareknowntocontainall four alkaloids, whilematureones
contain only arecoline.
5
The widespread consumption and use of
arecanut or its products raises concerns about the nutritional and
Cprior touseandshouldbefreshlypreparedbymixing0.2mol L
1
acetate buffer (pH 3.6), 10 mmol L
1
TPTZ in 40 mmol L
1
HCl and
20mmol L
1
FeCl
3
ina10:1:1ratio. Astandardcurvepreparedusing
Trolox in the range 50500 mol L
1
gave a regression equation
Y =0.001X (R
2
=0.997), which correlated the absorbance at 593
nm (Y) and Trolox (X). The nal results were expressed as Trolox
equivalent antioxidant capacity (TEAC) per gram of sample.
Determination of ABTS radical scavenging activity
Antioxidant power of arecanut extract was assessed according to
the ABTS assay described by Abraham and Mathew.
17
The ABTS
radical cations (ABTS
.+
) was producedbyreacting7mmol L
1
stock
solution of ABTS with 2.45 m mol L
1
potassium persulfate (nal
concentration) and allowed to stand in the dark for at least 16 h at
27 2
(A
S
A
1
) / (A
0
A
1
)
100
where, A
S
, A
1
and A
0
are the absorbance of the sample, control
solution containing 1,10-phenanthroline, FeSO
4
and H
2
O
2
and
blank solution containing 1,10-phenanthroline and FeSO
4
.
Determination of arecoline content by HPLC
Arecolinecontent of theextract was determinedbyHPLCusingthe
method described by Aromdee et al.,
19
with some modication.
The Jasco HPLC system tted with UV-visible detector (UV-1575)
at 216 nm was used at an isocratic ow of 0.8 mL min
1
at
45
C. The detection of analytes was accomplished with a C
18
column (Spherisorb ODS2, 5 m, 4.6 250 mm analytical column,
Waters, Milford, MA, USA). Themobilephasecompositionusedwas
potassium dihydrogen phosphate (10 g L
1
), phosphoric acid (3.5
mL L
1
), triethylamine (8 mL L
1
) and acetonitrile (12 mL L
1
), and
was ltered through 0.22 before use. The pHof the mobile phase
was 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 correlating
the area under the peak (Y) and arecoline (X) was Y =60326X
(R
2
=0.998).
Identication of polyphenols by HPLC
The analysis of polyphenols was performed using HPLC (Flexar
600, PerkinElmer, Waltham, MA, USA) with a Hypersil C
18
column
(Brownlee Analytical C
18
, 4.6 m, 4.6 150 mmanalytical column)
of length 250 mm using a UV-visible detector at 280 nm. The col-
umn was equilibrated with acetonitrile (B) 0.1%orthophosphoric
acid (A). The column was mounted on a PerkinElmer Flexar 200
pumpHPLCchromatographequippedwith20Lloopinjector. The
gradient analysis was carried out for 33 min at 25
C. The multilin-
ear gradient beganwith100%Aupto3min, 90%A+10%Bfrom3
wileyonlinelibrary.com/jsfa c 2013 Society of Chemical Industry J Sci Food Agric (2013)
Separation of polyphenols and arecoline from areca nut www.soci.org
Epicatechin
Catechin
Figure 2. HPLC chromatogram of arecanut crude extract.
to 6.5 min, 75%A+25%B from6.5 to 14 min, 45%A=55%B from
14 to 25 min, and nally with 10% A+90% B from 25 to 33 min.
Identication of arecanut constituents using LCMS
A Varian LCMS-500L systemequipped with ion trap mass analyser
having a mass range from 20 to 2000 amu, photodiode array
detector (PDA), autoinjector, and LC binary pump coupled online
withanMS-500mass spectrometer (VarianInc., PaloAlto, CA, USA).
Electrospray ionization (ESI) was operated in the negative mode.
Ion spray voltage was set at 4000 V and orice voltage 60 V.
HPLC separation was carried out on a narrow-bore reversed-phase
column (C
18
). The column was connected with a mass interface
via fused-silica capillary tubing (length 100 cm, 100 m i.d.). The
sample was injected with a rotary valve tted with 20 L sample
loop. Elution was achieved using the mobile phase acetonitrile
(A):water (B). Gradient analysis was carried out for 15 min. The
gradient 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 nally with 100%
A+0% B from 10 to 15 min.
Statistical analysis
All data were expressed as means standard error of triplicate
measurements and analysed by SPSS for Windows (version
16.0, SPSS Inc., Chicago. IL. USA). One-way analysis of variance
(ANOVA): post hoc multiple comparisons were carried out to test
any signicant differences between solvents used, solvent:water
ratio, pH, substrate concentration and extraction time. Statistical
comparisons between variables (e.g. TPC, TTC, arecoline and
antioxidant activity) were performed using the least square
differential method (LSD). Differences were considered signicant
at P <0.05.
RESULTS ANDDISCUSSION
Effect of solvent
Various polar and non-polar solvents such as water, methanol,
ethanol, n-butanol, acetone, chloroform, ethyl acetate and hexane
were used for the extraction of TPC, TTC and arecoline from dried
arecanut powder. Among these, acetone resulted in maximum
extraction of polyphenols (218.29 mg GAE g
1
of sample) and
tannin (53.34 mg CE g
1
of sample). Interestingly, acetone also
gave minimum extraction of arecoline (2.30 mg g
1
of sample),
whereas methanol resulted in maximum extraction followed by
ethanol (Table 1). These results indicate arecoline to be soluble
in water and alcohol and are in agreement with those of
Chiang et al.
20
Naczk and Shahidi
21
reported the extraction of
phenolic compounds to depend on the solubility and polarity of
solvents. Furthermore, acetone also showed a maximum ratio of
TPC:arecoline, which again signied the extraction of maximum
polyphenols with minimum arecoline.
Besides TPC, antioxidant assays suchas ABTS, FRAPandhydroxyl
radical scavenging activity were carried out to determine the
antioxidant potency of extracts obtained. Maximum ABTS (422.73
mmol L
1
TEAC g
1
of sample), FRAP (947.50 mmol L
1
TEAC
g
1
of sample) and hydroxyl radical scavenging activity (28.18%)
was also seen with the acetone extract. This was followed by
methanol, ethanol and water, which were parallel to the phenolic
content (Table 1). Pan et al.
22
reported a correlation between
phenolic compounds and antioxidant activities. In our work too,
a correlation between the content of phenolic compound and
antioxidant activity was observed. Since the solvent inuences
the extraction of polyphenols, it also had a similar effect on the
antioxidant prole.
Further extraction of phenolic compounds was carried out
using acetone with varying proportions of water. It is evident
from Table 2 that 80% acetone gave the highest TPC (297.30
mg GAE g
1
of sample), and TTC (68.53 mg CE g
1
of sample)
with a concomitant increase in arecoline (3.43 mg g
1
of sample),
which can be accounted for by the presence of water in the
solvent mixture. The phenolic content decreased at a water
content above 20%, which can be attributed to lower solubility
of phenolics in water. Alcohol water mixtures are known to be
more efcient than the corresponding mono-component solvent
system in extracting phenolic constituents.
23
The maximum ratio
of TPC:arecoline was observed in 80%acetone. It is observed from
Table 2 that ABTS, FRAP and hydroxyl radical scavenging activity
decreased with an increased proportion of water content in the
acetone:water blend and maximum activity was observed in the
solvent blend containing 80% acetone. This may be due to a
change in solvent polarity, which helps to dissolve the selected
group of antioxidant compounds and inuences the antioxidant
activity.
24
The antioxidant activity showed signicant correlation
with TPC. The maximum ABTS (429.80 TEAC g
1
of sample), FRAP
(997.28 mmol L
1
TEAC g
1
of sample), and hydroxyl radical
scavenging activity (53.34%) and phenolic content was observed
in solvent containing 80%acetone. Yilmaz and Toledo
25
found the
phenolic content of ethanolic extract from grape seed to increase
with increase in water from0 to 30%in the mixture and decreased
with a further increase in water. Similar results were reported
by Cacace and Mazza
26
on blackcurrant using aqueous ethanol
J Sci Food Agric (2013) c 2013 Society of Chemical Industry wileyonlinelibrary.com/jsfa
www.soci.org Y V Chavan, R S Singhal
5 10 15 20 25 minutes
10
20
30
40
50
60
70
80
kCounts -CRUDE EXTRACT 5-25-2012 12-30-05 PM.XMS 156.0+247.0+207.0+149.0 (100:400) Filtered
100:400
1A
1B
100 150 200 250 300 350 400 m/z
R.Match: 578, F.Match: 426
0
10
20
30
kCounts
Search
F F F
105.1
5051
133.2
3678
149.1
11190
152.2
2095
156.2
37037
172.2
7659
203.2
14992
207.2
6882
225.3
5309 245.3
2697
291.2
2824
Spectrum 1A
1.836 min, Scan: 125, 100:400, Ion: 60198 us, RIC: 400566 BP: 156.2 (37037=100%), -crude extract 5-25-2012 12-30-05 pm.xms
0
5
10
15
kCounts
Match
F
107.1
3200
123.1
14346
127.1
6001
133.2
1399
139.1
20603
147.2
6821
152.1
1278
173.1
3776 193.3
2262
207.3
11275
215.3
3226 233.3
1594
247.3
6486
259.2
2371
287.3
4194
Spectrum 1B
7.301 min, Scan: 495, 100:400, Ion: 115371 us, RIC: 310540 BP: 139.1 (20603=100%), -crude extract 5-25-2012 12-30-05 pm.xms
N O O
CH
3
H
3
C
(A)
Figure 3a. HPLC-ESI-MS of the sample extracted from arecanut: ion chromatogram extracted at m/z value corresponding to the M+H ion of arecoline.
and found 70% ethanol to be the best for extraction of phenolic
compound. Hence80%acetonewas usedfor further studies. Wang
and Hwang
27
also used 80% acetone for extraction of phenolic
compound from betel quid using different extraction protocols,
although they did not estimate the content of arecoline or the
antioxidant activity of betel nut. Han et al.
28
extracted TPC from
betel nut with 70% ethanol using ultrasound-assisted extraction.
Effect of extraction conditions
Arecanut powder was extracted with 80% aqueous acetone at
different pH values and analysed for TPC, TTC, arecoline and
antioxidant activity. The extraction of TPC and TTC increased with
an increase in pH from 2 to 6 and decreased sharply above pH 6
(Table 3), while that of arecoline also increased with an increase
in pH from 2 to 10. A pH of 4 showed good extraction of TPC
(315.07 mg GAE g
1
sample) and of TTC (81.15 mg CE g
1
sample)
with minimum arecoline (1.24 mg g
1
). The variation observed in
phenolic content with change in pH may be accounted for by the
selective solubility characteristics of some polyphenols at these
pH values.
29
The ratio of TPC:arecoline also varied with pH and
was found to be maximal at pH 2. Since the total phenolic content
was lower at pH 2, TPC:arecoline ratio at pH 4 was considered to
be the optimum which resulted in higher phenolic content with a
lower amount of arecoline.
In addition to phenolic content, extract was analysed for
antioxidant potency. The antioxidant capacity of arecanut extract
was exhibited by high ABTS (458.82 mmol L
1
TEAC g
1
of
sample), FRAP (1010.14 mmol L
1
TEAC g
1
of sample) and
hydroxyl radical scavenging activity (49.78%) at pH 4 and can
positively be correlated with phenolic content (Table 3). A change
in colour of the extract was also observed with an increase in pH.
This could be due to precipitation of the coloured pigment
wileyonlinelibrary.com/jsfa c 2013 Society of Chemical Industry J Sci Food Agric (2013)
Separation of polyphenols and arecoline from areca nut www.soci.org
5 10 15 20 25 minutes
0
25
50
75
kCounts -CRUDE EXTRACT 5-25-2012 12-30-05 PM.XMS 100:400 (-) Filtered
100:400 (-)
1A
1B
100 150 200 250 300 350 400
m/z
R.Match: 116, F.Match: 6
0
1
2
3
kCounts
Search
124.9
2862
136.9
531
160.9
1702
176.9
558
203.0
600
221.1
608
245.1
1441
255.2
339
271.1
214
287.1
605
289.0
3874
Spectrum 1A
6.997 min, Scan: 472, 100:400 (-), Ion: 141755 us, RIC: 23754 BP: 289.0 (3874=100%), -crude extract 5-25-2012 12-30-05 pm.xms
0.0
2.5
5.0
7.5
10.0
kCounts
Match
207.1
12590
208.1
1461
227.1
940
255.1
862
325.1
1770
Spectrum 1B
11.937 min, Scan: 816, 100:400 (-), Ion: 206675 us, RIC: 24301 BP: 207.1 (12590=100%), -crude extract 5-25-2012 12-30-05 pm.xms
Catechin: 289 (M+H)
HO
HO
O
OH
OH
OH
(B)
Figure 3b. HPLC-ESI-MS of the sample extracted from arecanut: ion chromatogram extracted at m/z value corresponding to the M+H ion of catechin.
or alteration in chromophoric characteristics under alkaline
conditions.
30
In these sets of experiments too, the extract of arecanut
showed positive correlation between antioxidant activity (ABTS,
FRAP and hydroxyl radical scavenging activity) and polyphenol
concentration (Fig. 1A), which is in agreement with previous
reports for tea polyphenols and antioxidant activity.
31
The
corresponding correlation coefcients were 0.984 for ABTS
(y =0.157x +408.6), 0.99 for FRAP (y =9.415x 692.45) and 0.978
for hydroxyl scavenging activity (y =0.073x +27.78).
The effect of sample concentration (614%) on extraction of
TPC, TTC and arecoline showed 14% to extract maximum TPC
(396.22 mg GAE g
1
of sample), TTC (104.71 mg CE g
1
of sample)
and arecoline content (2.58 0.01 mg g
1
of sample) (Table 4).
A signicant difference was observed in TPC at 10%, 12% and
14% w/v of sample but, with an increase in the concentration of
arecanut, extraction of arecoline also increased. The TPC:arecoline
ratio also increased with an increase in concentration and
maximum TPC:arecoline ratio (239.01) was observed with 10%
w/v. Hence 10% concentration was optimized for further study.
Furthermore, ABTS (458.62 mmol L
1
g
1
of sample), FRAP
(1011.44 mmol L
1
TEAC g
1
of sample) and percent hydroxyl
radical scavenging activity (52.77%) at 10%w/v was observed. The
trend of antioxidant activity was similar to that of TPC. This may
be due to the ability of antioxidants to scavenge certain radicals
with higher concentration.
32
To evaluate the experimental results,
attempts were made tocorrelate the content of polyphenols inthe
extracts with their antioxidant activity (Fig. 1B). TEAC was found
to be concentration-dependent and agrees with a previous work
on linear correlation between TPC and antioxidant activity.
33
The
ABTS, FRAP and hydroxyl scavenging activity also showed good
correlation with TPC. The correlation coefcients were 0.916 for
FRAP (y =3.089 378.54), 0.922 for ABTS (y =1.618x 122.8) and
0.967for percent hydroxyl scavengingactivity(y =0.210x 19.45).
J Sci Food Agric (2013) c 2013 Society of Chemical Industry wileyonlinelibrary.com/jsfa
www.soci.org Y V Chavan, R S Singhal
75 100 125 150 175 200 m/z
0
5
10
15
20
25
Counts
171.9
11
178.8
26
199.0
6
Spectrum 1A
0.233 min, Scan: 17,199.0>75:209 (-) [1.20V], Ion: 250000 us, RIC: 43 BP: 178.8 (26=100%), sample-199-msms.xms
(a) Arecoline N oxide: 171
(b) Syringic acid or
Arecaidinylglycine
199 (M+H)
N
+
-O
CH3
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 sample
was kept on an orbital shaker at 180 rpm, pH 4 and 10% sample
concentration. Samples were drawn at regular intervals of 30
min and the extract was analysed for TPC, TTC, arecoline and
antioxidant activity. The extraction of TPC and TTC from arecanut
increased with an increase in extraction time up to 90 min and was
signicantly altered after 120 and 150 min of extraction (Table 5).
Optimum extraction of TPC and TTC with an adequate amount
of arecoline was observed at 90 min. The amount of arecoline
also increased with an increase in extraction time, and maximum
TPC:arecoline ratiowas foundat 60 min. Lapornik et al.
34
foundthe
yield of grape polyphenols to increase with time, while in the case
of alcohol extracts it strongly increased with extraction time up to
a 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 found
to be optimal at 90 min. Activity of ABTS (504.71 mmol L
1
per gram of sample), reducing power of FRAP (1043.33 mmol
L
1
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 for
further studies. This may be due to the degradation or oxidation
of phenolics on prolonged extraction. Perva-Uzunalic et al.
35
reported on extraction of catechin with water from green tea and
obtained maximumextraction efciency after 20 min. Yang et al.
36
studied various extraction parameters on Phyllanthus emblica L.
for extraction of polyphenols and among all parameters found
solvent concentration (75% ethanol) and extraction time (45 min)
to have a signicant effect on polyphenol extraction.
Identication of arecanut bioconstituents by HPLC and LC-MS
Polyphenols like catechin and epicatechin were tentatively
identied based on HPLC and comparison of their electronic
absorption spectra with authentic standards. For identication
of polyphenolic compounds, HPLC was used with a UV detector
at 280 nm. Two major peaks eluting at 4.5 and 7.11 min in
a total run time of 33 min were seen in the chromatogram
of 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 of
either or both of these compounds conrmed the presence
of catechin (4.5 min) and epicatechin (7.14 min) in arecanut
extract. For further conrmation, LC-MS was performed with the
extract.
Mass spectrometry is a highly sensitive technique requiringvery
small amounts of material (typically 1 g) for analysis. The process
of elucidating the mass and the structure of a substance can be
expedited by using a high-performance mass spectrometer. Using
ESI mass spectrometry, whichproducedprotonatedmolecular ions
with minimal or no fragmentation, pseudo-molecular ions of each
eluting species can be readily produced. ESI mass spectrometry
is superior to others in this respect since structurally signicant
information can be elicited by collisional activation of the eluting
species in the vacuum interface of the ESI source.
The presence of unchanged arecoline in crude extract was
determined from the occurrence of an ion of [M+H]
+
=156
m/z which gave a match for C
8
H
13
NO
2
(Fig. 3a). The presence of
catechin and epicatechin was also identied by the occurrence
of [M+H]
+
=289.0 m/z and which gave a match for C
9
H
14
N
2
O
3
(Fig. 3b). Giri et al.
37
reportedthe presence of arecoline Noxide and
arecaidinylglycine from the presence of an ion of [M+H]
+
=172
m/z (C
8
H
14
NO
3
) and [M+H]
+
=199.1 m/z, respectively, during
their study on a metabolic approach to arecanut alkaloids, namely
arecoline and arecaidine, in mouse urine
.
These compounds
are probably present in arecanut extract as metabolic products
due to indigenous plant enzymes. The peak corresponding to
[M+H]
+
=199.1m/z mayalsobeattributedtosyringicacid, which
has been reported by Zhang et al.
38
at the same [M+H]
+
=199.1
m/z. However, the major peak observed in Fig. 3c could not be
identied. The presence of acid in the mobile phase is essential
for complete separation and elution of these analytes.
37
Besides
these, 13 other compounds which could not be identied were
also obtained.
In conclusion, maximum extraction of polyphenols with mini-
mum extraction of arecoline was obtained using 80% acetone of
pH 4 with 10% arecanut concentration for 90 min. The extract so
obtained gave a TPC of 407.47 mg GAE g
1
of sample, a TTC of
101.82 mg CE g
1
of sample, and arecoline at 1.73 mg g
1
of sam-
ple. The antioxidant activities of the extracts, as seen from ABTS,
FRAP and hydroxyl scavenging assays, correlated with the total
phenolics. Separation and identication of individual components
in arecanut was conrmed using HPLC and LC-MS. The presence
of catechin, epicatechin andarecoline in arecanut extract was con-
rmed fromHPLC and LC-MS chromatograms. These results could
be useful in obtaining arecoline-free extraction fromarecanut and
using the otherwise toxic bioresource for benecial purposes. Fur-
ther work on minimizing the extraction of arecoline is in progress.
ACKNOWLEDGEMENT
We are thankful tothe University Grants Commission, Government
of India, New Delhi, for providing a fellowship to the rst author
and nancial support for the project.
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