10 International Journal of Food Science and Technology 2006, 41 (Supplement 1), 10–14

Original article
Piper betle: a potential natural antioxidant

Lakshmi Arambewela*, Menuka Arawwawala & Damisha Rajapaksa

Industrial Technology Institute, Bauddhaloka Mawatha, Colombo 7, Sri Lanka
(Received 27 April 2005; Accepted in revised form 18 April 2006)

Summary Piper betle Linn. (Family: Piperaceae) is a common plant cultivated in Asian countries. The aim of this study
was to evaluate the antioxidant potential and possible applications of P. betle leaves grown in Sri Lanka.
This was carried out using P. betle cold ethanolic extract (CEE), hot water extract (HWE) and essential oil
(EO). The initial free radical scavenging activity of CEE was higher than that of butylated hydroxyl toluene
(BHT). Further, antioxidant activity of CEE, EO and HWE did not significantly deviate from the initial
antioxidant activity up to 12 months. However, at elevated temperature (200 C) antioxidant activity was
significantly reduced (EC50 values of CEE, EO and BHT increased by fourfold and HWE by threefold)
compared to their initial values. The reduction of antioxidant activity of CEE was lesser than that of BHT.
Peroxide values (PV) were significantly lower in CEE-incorporated coconut and palm oil samples compared
to that of BHT-treated samples. Moreover, CEE extended the shelf-life of potato chips and increased the
stability of Aloe gel.
Keywords Antioxidant activity, Piper betle, rancidity.

Sri Lankan betel. Further, very few investigations on the

Introduction
Piper betle Linn. (Family: Piperaceae) is a perennial
dioecious, semiwoody climber. Stems were strongly
swollen at the nodes, and papillose when young. Leaves
were alternate, simple and yellowish green to bright
green in colour. Piper betle is cultivated in Sri Lanka,
India, Malaysia, Indonesia, Philippine Islands and East
Africa (Jayaweera, 1982; Dassanayake & Fosberg,
1987). More than 12 P. betle cultivars are reported in
Sri Lanka (Ratnasoma & Senevirathna, 1995) and
except cultivar called Malabulath which is not used for
chewing, other cultivars constitute ‘commercial betel’ of
Sri Lanka. According to Kumaratunga (2003), chemical
constituents and their relative proportions in essential
oil (EO) of ‘commercial betel’ of Sri Lanka are different
from that of other countries. The EO was characterised
by high content of safrol. In addition, eugenol, allyl
diacetoxy benzene and chavibitol acetate were identified
as major constituents of the Sri Lankan betel leaves.
Piper betle leaves are credited with many medicinal prop-
erties such as digestive, stimulative, carminative and
aphrodisiac (Anon., 1992). However, Sri Lankan P. betle
inhibits male sexual behaviour in rats and possesses
antiaphrodisiac activity (Ratnasooriya & Premakumara,
1996) indicating the differences in biological activities of
*Correspondent: Fax: +94 11 2686 567;
e-mail: larambewela@yahoo.co.uk
activities of P. betle grown in Sri Lanka are reported
except the experiments on antifertility effects of male
rats (Ratnasooriya & Premakumara, 1997), antimotility
effects on washed human spermatozoa (Ratnasooriya
et al., 1990) and antimicrobial activities (Kumaratunga,
2003). Piper betle grown in other countries are shown to
possess antimicrobial (Tewari & Nayak, 1991), gastro-
protective (Majumdar et al., 2003), wound healing
(Santhanam & Nagarajan, 1990), hepatoprotective
(Saravanan et al., 2002) and antioxidant (Choudhary
& Kale, 2002; Saravanan et al., 2002; Santhakumari
et al., 2003) activities. Therefore, investigations on the
antioxidant potential of P. betle grown in Sri Lanka
were undertaken because of the biological and chemical
differences of the plant. The antioxidant activities of
P. betle cold ethanolic extract (CEE), hot water extract
(HWE) and EO were determined using 2,2-diphenyl-1-
picrylhydrazyl (DPPH) scavenging activity assays. In
addition, possible applications of P. betle as a natural
antioxidant were also investigated.
Materials and methods
Plant material
Piper betle leaves were purchased from main vegetable
markets in Western province of Sri Lanka in March
2003. The leaves were identified and authenticated

doi:10.1111/j.1365-2621.2006.01227.x
 2006 Institute of Food Science and Technology Trust Fund

by curator of National Herbarium, Royal Botanical
Gardens, Peradeniya, Sri Lanka. A voucher specimen
(PS 01) was deposited in the Industrial Technology
Institute, Colombo 7, Sri Lanka.
Preparation of hot water extract
Piper betle leaves were air dried for 3–5 days in the
shade and cut into small pieces. Five hundred grams was
boiled with 2.5 L of distilled water for 4 h. The HWE
was concentrated under vacuum, freeze dried (yield
26.2% w/w dry weight basis) and stored at 4 C until
use.
Preparation of cold ethanolic extract
Piper betle leaves were air dried for 3–5 days in the
shade and cut into small pieces. Five hundred grams was
macerated with ethanol (80% v/v) and kept for 48 h at
room temperature (28–30 C). The extract was filtered
and evaporated to dryness under reduced pressure at
50 C (yield 15.6% w/w dry weight basis) and stored at
4 C until use.
Preparation of essential oil
Piper betle leaves were air dried for 3–5 days in the
shade and cut into small pieces. Five hundred grams was
placed in a 3 L round bottom flask with distilled water
and hydrodistillation was carried out for 4 h using a
clevenger arm. The volatile oil was collected and added
sufficient amount of anhydrous sodium sulphate to
remove any water droplets present in the oil. Finally, oil
was stored at 4 C until use (yield 3.3% w/w dry weight
basis).
Phytochemical screening of HWE and CEE
Extracts were subjected for qualitative testing for
polyphenols, alkaloids, steroids, saponins and tannins
as described by Farnsworth (1996).
Determination of antioxidant activity of P. betle
Initial antioxidant activity
The antioxidant activity was determined by measuring
the remaining concentration of DPPHÆ as described by
Singh et al. (2002). In this assay, known concentrations
of (0–50 lg mL)1) CEE, EO, HWE and butylated
hydroxyl toluene (BHT) were taken into different test
tubes. The volume was adjusted to 1 mL by adding
methanol. Five millilitres of methanolic solution of
DPPHÆ (2 mg/100 ml methanol) was added to these
tubes and shaken vigorously. The tubes were allowed to
stand at room temperature for 20 min and the absorb-
ance was measured at 517 nm (Shimadzu UV-160, Co.
 2006 Institute of Food Science and Technology Trust Fund
Piper betle: a potential natural antioxidant Arambewela et al. 11
Ltd, Japan). Control was prepared as above by adding
methanol instead of extracts. BHT served as the positive
control. This experiment was repeated in triplicates.
The percentage of remaining DPPHÆ (% DPPHREM )
of each concentration was calculated as follows:
½DPPH T
%DPPHREM ¼  100
½DPPH C
where T is the experimental duration time (20 min) and
C is the control. EC50 value is the effective concentration
at which DPPHÆ radicals were scavenged by 50% and
calculated by plotting the % DPPHREM vs. the concen-
trations of extract used.
Antioxidant activity with time
After recording the initial antioxidant activity P. betle
extracts were stored at room temperature (28–30 C).
This experiment was repeated 9 and 12 months after the
initial experiment using same betel samples.
Antioxidant activity at elevated temperature (200 C)
Freshly prepared CEE, EO, HWE and BHT were kept
in an oven maintained at 200 C for 45 min and the
antioxidant activity was determined using DPPHÆ scav-
enging assay.
Determination of rancidity in coconut oil samples
incorporated with P. betle extracts
Coconut oil was added to 15 cleaned and dried glass
bottles (25 mL per bottle). They were randomly separ-
ated into five groups (n ¼ 3) and oil samples in groups 1,
2, 3 and 4 were incorporated with CEE, EO, HWE and
BHT (0.1 mg of each extract and/or BHT in 1 mL of oil)
respectively. The remaining oil samples served as the
control. All the bottles containing oil samples were
sealed, well mixed and kept in an incubator maintained at
50 C for 2 weeks. Peroxide value (PV) was measured in
these samples according to the Association of Analytical
Chemists (AOAC) (1995) method at regular intervals
(weekly) up to 2 weeks. Peroxide value is a measurement
which indicates the degree of rancidity in fats or oils.
Determination of rancidity in palm oil samples
incorporated with P. betle extracts
Same procedure was followed as described previously
using palm oil instead of coconut oil.
Determination of shelf-life of potato chips using cold
ethanolic extract of P. betle
Coconut oil was taken from the same oil container used
previously and poured into three frying pans (250 mL
per frying pan). CEE (0.2 mg/1 mL) and BHT (0.2 mg/
1 mL) were incorporated separately into two of the oil
International Journal of Food Science and Technology 2006, 41 (Supplement 1), 10–14
12 Piper betle: a potential natural antioxidant Arambewela et al.
samples and mixed well. The remaining oil sample
served as the control.
Potatoes were washed, peeled and cut into equal
pieces and randomly separated into three portions
(400 g per portion). The two portions of potato pieces
were deep fried to an oven yellow brown colour in
coconut oil which was incorporated with CEE and BHT
respectively. The remaining portions of potato pieces
were deep fried in coconut oil which was not mixed
either with CEE or BHT. The frying process was carried
out maintaining the temperature constant for 12 min.
To submerge the potato chips in the oil a frying basket
was used and after completing the frying, basket with
fried potato pieces was removed and allowed to stand
for 2 min for draining. Thereafter pieces were placed on
a filter sheet for further draining and cooling. Finally,
potato pieces were packed (100 g per bag) in polypro-
pylene bags and sealed. These were kept in an incubator
maintained at 42 C.
The coconut oil absorbed to potato pieces was
extracted to pet ether (60–80 C) using soxhlet extrac-
tion apparatus at regular intervals (3 weeks). Pet ether
was evaporated and the oil was subjected to determin-
ation of PV according to the AOAC (1995) method.
Effect of cold ethanolic extract on stability of Aloe gel
Aloe vera leaves were washed, cleaned and hung from
one end to remove the mucilage. Then the leaves were
peeled and gel was separated. Aloe gel was filled into a
glass jar and heated for 2 min at 70 C. Thereafter,
ascorbic acid 0.3%, sodium benzoate 0.1% and sodium
metabisulphite 0.2% were added and mixed well to
dissolve completely. Finally, bottles were sealed and
kept at room temperature (28–30 C). The colour of the
Aloe gel was observed every day and the time duration
(in terms of days) for discoloration of the gel was
recorded. The same procedure was carried out using
0.3% of CEE instead of ascorbic acid.
Statistical analysis
Statistical comparisons were made using one-way anova
followed by Tukey’s family error test. A P-value £0.05
was considered as significant.
Results and discussion
The extracts obtained from the leaves of P. betle had
profound antioxidant activity as judged by DPPHÆ
scavenging assay (Table 1). DPPHÆ scavenging assay is
one of the simple techniques used to measure the ability
of antioxidants to intercept free radicals (Navarro et al.,
1993). The scavenging effects of P. betle extracts on
DPPHÆ radicals decreased in the following order:
CEE > EO > HWE. Further, free radical scavenging
International Journal of Food Science and Technology 2006, 41 (Supplement 1), 10–14
Æ
Table 1 DPPH free radical scavenging activities of P. betle extracts
with time (n ¼ 6; mean ± SEM)
EC50 (lg mL)1)
Sample Initial After 9 months After 12 months
a a
CEE 6.23 ± 0.12 6.83 ± 0.16 7.00 ± 0.09a
EO 12.66 ± 0.07b 12.83 ± 0.17b 13.10 ± 0.14b
HWE 17.23 ± 0.12c 17.30 ± 0.20c 17.56 ± 0.15c
BHT 8.33 ± 0.07d 8.40 ± 0.09d 8.60 ± 0.05d
Values with the different superscripts are significantly different (P < 0.05)
according to the ANOVA and Tukey’s pairwise comparison test.
effect of CEE was higher than that of synthetic antioxid-
ant (BHT). Antioxidants react with the free radical
DPPHÆ and convert it to 1,1-diphenyl-2-picrylhydrazine
by donating a hydrogen (Son & Lewis, 2002).
The change in absorbance produced by this reaction
was used to test the ability of P. betle to act as a free
radical scavenger. Therefore, the above sequence in the
DPPHÆ radical scavenging activity indicates the hydro-
gen donating ability of betel extracts. According to the
literature (Sherwin, 1990; Jones, 1995), synthetic phe-
nolic antioxidants such as BHT and BHA also have
similar mode of action. Phenols are powerful antioxi-
dants (Singh et al., 2002). Phytochemical screening
revealed the presence of polyphenols, alkaloids, steroids,
saponins and tannins in both extracts. Therefore,
antioxidant activity of P. betle may be due to the
phenolic compounds that are present in the plant. A
number of plant alkaloids (Azam et al., 2003; Tachib-
ana et al., 2003) and flavonoids (Siddhuraju & Becker,
2003; De Sousa et al., 2004) have been shown to possess
antioxidant properties. As both alkaloids and flavonoids
are present in the extracts of P. betle, the involvement of
these constituents for the observed antioxidant potential
cannot be completely ruled out.
Interestingly, the antioxidant properties of P. betle
extracts including CEE, EO and HWE remained unal-
tered (Table 1) up to 12 months at room temperature.
This supports the use of the betel extracts as a natural
antioxidant in food industry. However, when P. betle
extracts were exposed to the elevated temperature
(200 C), the antioxidant properties were significantly
reduced (CEE and EO by fourfold; HWE by threefold).
Similar result was also evident with the synthetic
antioxidant BHT (by fourfold). Even after exposure to
the elevated temperature the antioxidant potential of
CEE was higher than that of BHT (Table 2). The
reduction of antioxidant activity of betel extracts under
the elevated temperature may be possibly due to the
change in the chemical constituents.
In this study P. betle extracts were incorporated into
both coconut and palm oil separately and rancidity in
terms of PV was determined. CEE had the best
 2006 Institute of Food Science and Technology Trust Fund
 Piper betle: a potential natural antioxidant Arambewela et al. 13

Æ
Table 2 DPPH free radical scavenging activities of P. betle extracts at
elevated temperature (n ¼ 6; mean ± SEM)
EC50 (lg mL)1)
At room temperature At elevated temperature
Sample (30 °C) (200 °C)
CEE 6.23 ± 0.12a 24.43
EO 12.66 ± 0.07b 50.26
HWE 17.23 ± 0.12c 48.96
BHT 8.33 ± 0.07d 32.30
Values with the different superscripts are significantly different (P < 0.05)
according to the ANOVA and Tukey’s pairwise comparison test.
capability of reducing the rancidity in coconut and palm
oil samples followed by EO, BHT and HWE respect-
ively. At the same time, P. betle extracts reduced the PVs
of the coconut oil more prominently than that of the
palm oil (Table 3). Lauric acid is the major component
(48.2%) in the coconut oil followed by caprylic acid
(7.6%), capric acid (7.3%), myristic acid (16.6%),
palmitic acid (8.0%), palmitoleic acid (1.0%), stearic
acid (3.8%), oleic acid (5.0%) and linoleic acid (2.5%)
(Weiss, 1983). Palm oil is similar to coconut oil but is
more unsaturated as seen from the following composi-
tion. More than 50% of palm oil is lauric acid (50.9%),
followed by caprylic acid (1.4%), capric acid (2.9%),
myristic acid (18.4%), palmitic acid (8.7%), stearic acid
(1.9%), oleic acid (14.6%) and linoleic acid (1.2%)
(Weiss, 1983). Polyunsaturated fatty acids are more
susceptible to oxidation than monounsaturated fatty
acids. Moreover, the greater number of double bonds in
the fatty acid, the more prone it is to peroxidation. Palm
oil is capable of being rancid faster than coconut oil as it
is more unsaturated. This may be the reason that betel
extracts retard the rancidity of palm oil less effectively.
Frying oil may undergo both oxidative and thermo-
lytic degradation. Oxidation products are classified as
volatile, monomeric or polymeric compounds. Some
volatile decomposition products are removed from the
oil by steam that evolves during frying of food. Other
reaction products may remain in oil as nonvolatile
decomposition products and some of these are absorbed
± 0.56e
to the food product (Perkins & Erickson, 1996).
± 0.47f
± 0.92f
Eventually these compounds get oxidised and rancidity
± 0.64g
occurs. Therefore, to avoid the oxidation, antioxidants
are incorporated into the frying oil. The lowest PV was
evident in coconut oil, which was extracted from the
potato chips that treated with CEE (Table 4). This is a
favourable indicator for CEE to be used as a natural
antioxidant in deep frying products. On the other hand,
in extruded products the oil is used at low temperatures
with no presence of water and the antioxidants can be
effective in increasing the shelf-life by delaying the onset
of rancidity. Therefore, better results can be obtained if
CEE is incorporated to an extruded product. Further
discoloration in Aloe gel occurred 22 days later in CEE-
treated sample than that of ascorbic acid-treated sample
(Table 5). This indicates the potential of CEE as a
stabiliser. All these observations support the possible
applications of P. betle extracts to use in food industry.
According to Arambewela et al. (2003) both extracts
were devoid of unacceptable side effects even after
following chronic administration at a dose of
1500 mg kg)1. There were no overt signs of toxicity,
hepatotoxicity (in terms of serum AST, ALT levels) or
renotoxicity (as judged by serum urea and creatinine
levels). In conclusion, this study demonstrated the
antioxidant activity and possible applications of ‘com-
mercial betel’ of Sri Lanka for the first time.
Table 4 The peroxide values (PVs) of the coconut oil extracted from
potato chips (n ¼ 3; mean ± SEM)

Table 3 The effects of P. betle extracts on peroxide values (PV) of the PV (mEq kg)1)
coconut oil and palm oil (n ¼ 3; mean ± SEM)
Time Oil extracted from Oil extracted from
PV (mEq kg)1) duration CEE (0.2 mg mL)1)- BHT (0.2 mg mL)1)-
(weeks) Control incorporated samples incorporated samples
Sample First week Second week
3 3.26 ± 0.10 1.22 ± 0.04* 1.60 ± 0.04*
Coconut oil (control) 4.26 ± 0.37 6.86 ± 0.19
6 6.34 ± 0.07 2.70 ± 0.05* 4.46 ± 0.07*
Treated coconut oil (0.1 mg mL)1)
9 13.93 ± 0.31 6.13 ± 0.16* 11.42 ± 0.47*
CEE 1.32 ± 0.06* (69.0%) 2.61 ± 0.02* (61.9%)
EO 1.44 ± 0.07* (66.2%) 3.39 ± 0.08* (50.6%) *Significant at P < 0.05 level compared with the control.
HWE 2.33 ± 0.03* (45.3%) 5.20 ± 0.18* (24.2%)
BHT 1.66 ± 0.05* (61.0%) 3.62 ± 0.04* (47.2%)
Table 5 Stability of Aloe gel (n ¼ 3)
Palm oil (control) 5.91 ± 0.12 12.93 ± 0.93
Treated palm oil (0.1 mg mL)1) Treatment Time duration to occur discoloration (days)
CEE 3.64 ± 0.07* (38.4%) 5.60 ± 0.49* (56.7%)
EO 4.46 ± 0.09* (24.5%) 7.83 ± 0.31* (39.4%) 0.3% CEE 47 ± 0*
HWE 5.03 ± 0.10* (14.9%) 10.13 ± 0.26* (21.6%) 0.3% Ascorbic acid 25 ± 0
BHT 4.59 ± 0.03* (22.3%) 8.52 ± 0.04* (34.1%)
*Significant at P < 0.05 level compared with the ascorbic acid treated
*Significant at P < 0.05 level compared to the control. sample.

 2006 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2006, 41 (Supplement 1), 10–14
14 Piper betle: a potential natural antioxidant Arambewela et al.

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Science Foundation for the Research Grant (SIDA (1L) Pp. 3–5. Sri Lanka: Department of Agriculture.
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International Journal of Food Science and Technology 2006, 41 (Supplement 1), 10–14  2006 Institute of Food Science and Technology Trust Fund