Food Anal.
Methods
DOI 10.1007/s12161-012-9470-y
TLCBioautography-Guided Isolation, HPTLC and GCMS-Assisted
Analysis of Bioactives of Piper betle Leaf Extract Obtained
from Various Extraction Techniques: In vitro Evaluation
of Phenolic Content, Antioxidant and Antimicrobial Activities
H. V. Annegowda & P. Y. Tan & M. N. Mordi &
S. Ramanathan & M. R. Hamdan & M. H. Sulaiman &
S. M. Mansor
Received: 19 April 2012 / Accepted: 3 July 2012
# Springer Science+Business Media, LLC 2012
Abstract The polyphenolic content, antioxidant and anti-
bacterial activities of the ethanolic extracts of Piper betle
leaf obtained from soxhlet (PBSx), sonication (PBSn) and
maceration (PBMn) extraction methods were studied. GC
MS analysis was carried out to determine the variation in the
phytoconstituents in these extracts. Whereas, thin-layer
chromatography (TLC)bioautography was conducted to
localise, separate, and identify antioxidants, and their
amount was determined by the newly developed high-
performance thin-layer chromatography (HPTLC) method.
The results of polyphenolic content, antioxidant assays and
antimicrobial assay showed that PBSn contained significant
amount of polyphenolics, antioxidant and antimicrobial ac-
tivity followed by PBMn and PBSx. Moreover, the obtained
antioxidant activity of PBSn was significant even in com-
parison with butylated hydroxytoluene (BHT) and commer-
cially available grape seed extract (GRSx). In addition, GC
MS analysis shown marked variations in the amount of the
phytoconstituents among all these extracts with PBSn con-
taining higher amount followed by PBMn and PBSx. TLC
bioautography resulted in the separation of three compounds
which are identified as eugenol, allylpyrocatechol, and
eugenyl acetate. The HPTLC densitometric determination
was also supported the results of antioxidant assays by
revealing the presence of higher amount of identified anti-
oxidants in PBSn followed by PBMn and PBSx. Since, P.
betle leaf extract has been used as one of the ingredients in
several herbal formulations, results of this study will not
H. V. Annegowda (*) : P. Y. Tan : M. N. Mordi :
S. Ramanathan : M. R. Hamdan : M. H. Sulaiman : S. M. Mansor
Centre for Drug Research, Universiti Sains Malaysia,
11800, Penang, Malaysia
e-mail: annegowdahv@gmail.com
only help the herbal industries in choosing the appropriate
extraction technique but also the developed HPTLC method
was simple, precise, sensitive and accurate hence can be
utilised for the routine quality control and standardisation of
formulations containing P. betle leaf extract.
Keywords P. betle . Sonication . TLCbioautography .
HPTLC validation . Eugenol . Allylpyrocatechol
Introduction
Medicinal herbs, vegetables and fruits are the important sour-
ces of bioactive compounds with therapeutical as well as
nutraceutical importance. Extraction is a critical step involved
in the discovery of bioactives. The nature and amount of
bioactive compounds isolated from the raw material mainly
depends on the type of extraction technique, temperature,
polarity of solvent employed and duration of extraction
(Nantitanon et al. 2010; Annegowda et al. 2012). The extraction
technique used must be able to retain the bioactive compounds
of commercial and therapeutical importance. Several isolation
techniques such as HPLC, column chromatography and TLC
bioautography are available for the separation of bioactive
compounds from the complex matrices. The advantage of quick
access about the activity as well as localisation of the bioactive
compounds present in the plants made TLCbioautographic
assay as an important tool in the search of novel lead com-
pounds from plants. (Simes-Pires et al. 2009).
The increased popularity of the natural products as a
source of medicine, nutraceuticals as well as cosmetics
along with their indiscriminate use resulted in the decline
in their quality and efficacy. Thus, several chromatographic
and spectroscopic analytical techniques such as HPLC, LC

MS, GCMS, TLC, HPTLC (high-performance thin-layer
chromatography), etc. are used to standardise these herbal
products to ensure their safety, quality and efficacy (Rajani
et al. 2001). Both HPLC and HPTLC fingerprinting analyt-
ical techniques are used to ensure the quality of plant mate-
rials or formulations containing it. The advantages of
rapidity, minimum sample cleanup, reduced sample con-
sumption, cost effectiveness and multiple sample analysis
at the same chromatographic condition made HPTLC as an
alternative to HPLC to evaluate the quality as well as to
standardise the herbal extracts or their formulations (Abou-
Donia et al. 2008).
Piper betle is a traditionally as well as economically
important plant which belongs to the family Piperaceae
and widely distributed in India, Sri Lanka, Malaysia, Indo-
nesia, Thailand, China and Philippines and subtropical
countries. Since ancient time, P. betle was not only used
traditionally for chewing purpose along with areca nut,
clove and cardamom but also as a source of medicine.
Traditionally, P. betle leaves were used to treat cold, cough
and also as a carminative, stimulant and digestive (Rawat et
al. 1989). Leaves of this plant possess anti-platelet (Jeng et
al. 2002), anti-allergic (Wirotesangthong et al. 2008), anti-
oxidant (Choudhary and Kale. 2002; Rathee et al. 2006),
antidiabetic (Arambewela et al. 2005a), anti-ulcer (Majumdar
et al. 2002) antimicrobial (Tappayuthpijarn et al. 1982), anti-
inflammatory (Pin et al. 2010), antifertility (Sarkar et al.
2000), analgesic (Arambewela et al. 2005b), wound healing
(Santhanam and Nagarjan 1990) and immunomodulatory ac-
tivities (Singh et al. 2009). Most of the pharmacological
activities like anti-ulcer, hepatoprotective, anti-carcinogenic,
anti-septic, thromboxane production and modulation of plate-
let aggregation possessed by this plant is predominately
through the antioxidant mechanism (Majumdar et al. 2002;
Jeng et al. 2002; Young et al. 2007). The important phenolic
constituents isolated from leaves of P. betle includes eugenol,
eugenol acetate, allylpyrocatechol, allylpyrocatechol monoa-
cetate, chavibetol acetate (Ramji et al. 2002), chavibetol
(Rathee et al. 2006), piper betol, piperol A and B (Zeng et
al. 1997), isoeugenol, methyl eugenol (Rawat et al. 1989) and
phytol (Jantan et al. 1994). Other essential oils isolated
includes, p-cymene, -terpineol, terpinyl acetate, caryophyl-
lenes (Sharma et al. 1983), 1,8 cineole, and -pinene, -
terpinene, -terpinene, -myrcene, camphene (Rawat et al.
1989; Jantan et al. 1994).
Currently, most of the research related to P. betle leaf
involves either isolation of phytoconstituents or evaluation
of pharmacological activities and very few studies regarding
the correlation of biological activity with the isolated com-
pounds are available (Ramji et al. 2002). Although, exten-
sive research is going on regarding the exploitation of P.
betle leaf has a potent antioxidant, no reports are available
regarding the influence of different extraction methods on
Food Anal. Methods
the polyphenolic content, antioxidant and antimicrobial ac-
tivity. Moreover, P. betle is one of the important ingredients
of several herbal formulations available in the market.
Hence, our main objective of the study is to locate, separate
and identify the antioxidants by TLCbioautography as well
as to develop and validate the HPTLC densitometric method
to quantify them which may serve as a quality control tool
for the standardisation of P. betle leaf extract and its for-
mulations; secondly, to evaluate and compare the phytocon-
stituents (from GCMS), polyphenolic content, antioxidant
and antibacterial activity of ethanolic extract of P. betle leaf
obtained from different extraction techniques.
Materials and Methods
Plant Material
Matured fresh leaves of P. betle were collected from the
campus of Universiti Sains Malaysia (USM), Penang
Malaysia, in September 2011. Damaged leaves were sorted
out from the collected leaves followed by washing with
running water to remove the dirt and adherent. These leaves
were then freeze dried and ground to obtain powder of 40
mesh size.
Chemicals and Reagents
2,2-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) dia-
mmonium salt (ABTS), potassium persulfate, 2,2-
diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-
triazine, FolinCiocalteu reagent, vanillin, sodium carbon-
ate, aluminium chloride hexahydrate, ferric chloride, p-
iodonitrotetrazolium, sodium acetate, sodium carbonate,
gallic acid, catechin, vanillin, butylated hydroxytoluene
(BHT), vitamin C, eugenol (EGL), eugenyl acetate and
allylpyrocatechol (APC) were purchased from Sigma Chem-
icals, Germany. MuellerHinton (MH) agar and broth were
from HiMedia Laboratories (Mumbai, India). Silica gel 60
GF254 TLC (HX083071), HPTLC plates (HX082452) and
solvents used in this study were of analytical and HPLC
grade and were purchased from E Merck KgaA (Darmstadt,
Germany). Ninety-six-well microtitre plates and lids were
purchased from Sterilin Ltd., UK.
Extraction Techniques
Three different extraction techniques, namely soxhlet, mac-
eration and sonication extraction techniques were employed
to obtain their respective extracts. All the extractions were
carried out with the same solid to solvent ratio of 50 g of the
ground leaf sample to 500 mL and ethanol (99.7 %, v/v) in
dark. Soxhlet extraction method was carried out at the
Food Anal. Methods
boiling point of ethanol (78 C) for 48 h whereas; macer-
ation was carried out at the room temperature for 3 days
with occasional stirring. Sonication was conducted at room
temperature for 60 min (20 min3) using BRANSON 5510
ultrasonic cleaner. The power level and sonication frequen-
cy were set at 135 W and 40 kHz, respectively. The liquid
extract obtained was then filtered through Whatman filter
paper (no. 1) and the marc left out from each extraction
method was resuspended in ethanol (2100 mL) for 5 min.
The filtrate obtained from the resuspended material was
combined with the earlier filtrate and then concentrated
using a rotary evaporator (Buchi Rotavapor R-215, Switzer-
land) followed by drying in a freeze dryer (LABCONCO,
FreeZone 6 Liter, USA) to obtain constant mass of respec-
tive soxhlet (PBSx), macerated (PBMn) and sonicated ex-
tract (PBSn). All the samples were kept in an air tight
container at 4 C until further analysis.
Determination of Total Phenolic, Total Flavonoids and Total
Tannins Content
The FolinCiocalteu method described by Singleton and
Rossi (1965) was followed to determine the total phenolic
content of P. betle leaf extracts obtained from different
extraction techniques, and results were expressed as milli-
gramme gallic acid equivalents/gramme dry extract. The
colorimetric method described by Sakanaka et al. (2005)
was followed to determine total flavonoid content present
in the extracts, and values were expressed as milligramme
catechin equivalents/gramme dry extract. Whereas, total
tannin content was determined by following the method of
Sun et al. (1998) and total tannins values were expressed as
milligramme CAE/gramme dry extract. All the measure-
ments were done in triplicate.
Determination of DPPH and ABTS Free Radical
Scavenging Activity
The method described by Blois (1958) and by Re et al.
(1999) was employed to evaluate DPPH and ABTS radicals
scavenging activities, respectively. These methods were
slightly modified and adapted to 96-well microtitre plate.
The stock solutions of P. betle leaf extract and standard were
twofold diluted using methanol to obtain varied concentra-
tions of 1.2580 and 0.62540 g/mL for DPPH and ABTS
assay, respectively. The reaction mixture without sample but
with the same amount of methanol (120 L) in the last well
of the 96-well plate served as control. Then, 120 L of
DPPH and ABTS solutions (1.00.02 units absorbance)
were added separately to the samples in each well and
shaken to ensure thorough mixing of reaction mixture. After
covering these plates with lid, incubated for 30 and 6 min
for DPPH and ABTS assays, respectively in dark at room
temperature. The decrease in the absorbance of reaction
mixture by antioxidant present in the sample was measured
at 517 and 734 nm for DPPH and ABTS radical assays
respectively, using Power wave X 340 microplate reader
(Bio-Tek instruments, Inc. USA). The percentage inhibition
of DPPH and ABTS radicals by tested samples was calcu-
lated using the following formula:
Percentage inhibition of DPPH radicals
Ao  A1 =Ao  100
where Ao is the absorbance of the control, and A1 is the
absorbance of samples. All the measurements were carried
out in triplicates.
Determination of Total Antioxidant Capacity and Ferric
Reducing Antioxidant Potency
The reducing ability of P. betle leaf extracts and standards
were evaluated following the total antioxidant capacity and
ferric reducing antioxidant potency (FRAP) assays. The
phosphomolybdenum method described by Prieto et al.
(1999) was followed to determine the reducing ability and
the total antioxidant capacity (TAC) values were expressed
as mg vitamin C equivalent antioxidant capacity (VCEAC)/
gramme dry extract. Similarly, the ferric ion reducing ability
of samples was evaluated using the modified method de-
scribed by Benzie and Strain (1996) and the FRAP values
were expressed as milligramme vitamin C equivalent anti-
oxidant value (milligrammes of VCEA)/gramme dry
extract.
Determination of Antibacterial Activity
Antibacterial activity of PBSx, PBSn and PBMn was ana-
lysed against three Gram-positive (Staphylococcus aureus
ATCC 29213, Bacillus cereus, Bacillus subtilis ATCC
19659) and four Gram-negative (Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella
pneumoniae ATCC 700603, Shigella) bacteria. The
microbes without ATCC number were obtained from School
of Biological Sciences, USM. The bacterial cell suspensions
were prepared in MH broth from 24-h culture and were
adjusted to the required concentration (McFarland 0.5) be-
fore the use.
The zone of inhibition (ZOI) and minimum inhibitory
concentration (MIC) of P. betle leaf extracts was evaluated
by agar diffusion method described by Bauer et al. (1966)
and Elof (1998), respectively. Sterile paper discs (6 mm
diameter, Oxoid, UK) impregnated with 20 L of crude
plant extracts (equivalent to 2 mg of the dried extract)
prepared in methanol was used for the determination of
ZOI whereas (0.312520 mg/mL) in DMSO was used for

the determination of MIC. Each experiment was carried out
in triplicate.
TLCBioautography-Assisted Separation of Antioxidants
TLCbioautography method not only helps to identify and
separate bioactive compounds but also to evaluate the contri-
bution of each compound within the extract for the found
pharmacological activity. Hence, TLC condition was opti-
mised and used for the TLCbioautography for the separation
of antioxidants from the complex mixture. Each antioxidant
compounds located by bioautography method on preparative
TLC plate were scrapped and carefully removed then dissolved
in ethyl acetate followed by centrifuging (10,000 rpm, Hermle
Centrifuge, Hermle Labortechnik GmBH, Germany) for
10 min. The procedure is repeated with fresh ethyl acetate until
there is no bleaching of DPPH by the supernatant solution. The
supernatant solution collected was then subjected for HPTLC
analysis and sprayed with sulphuric acid reagent and ferric
chloride reagent to identify the separated antioxidant
compounds.
HPTLC Method Validation and Quantification of Identified
Antioxidants
Stock solutions P. betle samples (10 mg/mL), eugenol, APC
and eugenyl acetate as well as three separated compounds
were prepared in methanol (2 mg/mL). Each 2 mL of stan-
dard solution prepared contained various concentration of
eugenol (125750 g/mL), APC (125750 g/mL) and
eugenyl acetate (2501,500 g/mL), respectively. Two
microlitres of each standard stock solution were applied
(band width: 4 mm and distance between track: 8 mm) on
silica gel 60 GF254 HPTLC plates (1020 mm) using Lino-
mat IV automated TLC applicator (Camag, Muttenz, Swit-
zerland) to obtain the concentration range of 2501,500 ng/
band of eugenol and APC and 5003,000 ng/band of
eugenyl acetate, respectively. With the help of the optimised
TLC condition the plate was developed to a distance of 7 cm
for 20 min then air-dried followed by observing under UV
and the images were documented. The amount of resolved
phytoconstituents were then quantified by HPTLC densito-
metric scanning using Camag TLC Scanner III in remis-
sionabsorption mode at 280 nm operated by Wincats
software (version 3.1) to obtain the Rf value and peak areas
of the HPTLC chromatogram.
HPTLC Method Validation
The HPTLC method developed was validated for instru-
mental precision, repeatability, linearity and accuracy. In-
strumental precision was determined by seven times
repeated scanning of the same spots of eugenol (800 ng/
Food Anal. Methods
band), APC (800 ng/band) and eugenyl acetate (1,600 ng/
band) and was expressed as percentage coefficient of vari-
ance (%CV). Repeatability of the method was confirmed by
analysing 800 ng/band of eugenol and APC and 1,600 ng/
band of eugenyl acetate individually (n 05) and was
expressed as %CV. Linearity of the method was determined
for the bioactives at the above-mentioned range. Intra-day
and inter-day precision of the method was determined by
analysing (n05) the aliquots of eugenol (800 ng/band), APC
(800 ng/band) and eugenyl acetate (1,600 ng/band) on the
same day and on three different days and the result was
expressed as %CV. Recovery study was performed to find
the accuracy of this HPTLC method by adding known
amount (25 mg) of eugenol, APC and eugenyl acetate to
1 g of the dried powder P. betle leaf followed by extraction
by sonication for 60 min and estimated as above. The
percentage recovery of eugenol, APC and eugenyl acetate
was also calculated.
Sample stock solutions were diluted appropriately so that
the concentration of these three bioactives applied in tripli-
cate (2 L) falls within their reference compounds calibra-
tion range. The compounds resolved on HPTLC plate with
antioxidant activity were located by spraying with 2.54 mM
DPPH reagent and observed in ordinary light for antioxidant
compounds after 30 min (Gu et al. 2009). Compounds with
antioxidant potency appeared as yellow bands against the
purple background. The antioxidant bands were confirmed
by comparing its Rf value with the Rf value obtained by
HPTLC densitometric determination. To evaluate the iden-
tity of antioxidants resolved in extracts as well as com-
pounds separated by preparative TLC, the UV spectrum of
each reference standard was overlaid with the corresponding
band in the sample track. Purity was also determined by
overlaying the absorption spectra at start, middle and end
position of the band resolved from extracts, separated com-
pounds with that of reference standards. The amount of
eugenol, APC and eugenyl acetate in sample solutions were
calculated using their respective calibration curves.
GCMS Analysis of Phytoconstituents in P. betle Extracts
P. betle leaf ethanolic extracts obtained from various extrac-
tion techniques were subjected to GCMS analysis to evaluate
their influence on extractability of various phytoconstituents.
Agilent gas chromatography mass spectrometer (GCMS
AGILENT 6890 N/5973I) coupled with time of flight detector
was used for the separation of various phytoconstituents using
HP-5 MS capillary column (0.25 mm30 m0.25-m film
thickness). The oven temperature was maintained at 70 C for
2 min and then gradually increased at 20 C/min to 305 C and
maintained at the same temperature for 1 min. Helium gas
with a flow rate of 1.2 mL/min was used as a carrier gas and
1 L of sample was injected for the GCMS analysis purpose.
Food Anal. Methods

The mass spectrometer was operated with the EI ion source
operating at 70 eV and acquisition range between 35 and 700
m/z, scan rate of one scan per second. Each resolved com-
pound was identified by Natural Institute of Standards and
Technology mass spectral library version 2.0 (NIST02 library,
NIST, USA).
Statistical Analysis
The results of each experiment was expressed as meansSD
(n 03). Analysis of variance was performed (one-way
ANOVA) and the significant differences between mean
values were determined by Tukeys pair-wise comparison
test at a level of significance of P<0.05 using SPSS 17
(SPSS Inc., USA). Pearson correlation was used to find
out the correlation between antioxidant activities and poly-
phenolic contents.
Results and Discussion
Extraction Yield, TPC, TFC and TTC of P. betle Leaf
Extracts
Several novel extraction techniques such as supercritical
fluid extraction, sonication, microwave-assisted and accel-
erated solvent extraction have been developed and
employed along with conventional extraction techniques
for the extraction of bioactive compounds and nutraceuticals
from plants (Wang and Weller 2006). But the preferred
extraction method should be simple, fast, economical and
able to retain the phytoconstituents of therapeutical and
nutraceuticals value. In this study, P. betel leaf was extracted
by soxhlet extraction, maceration and sonication using eth-
anol as extracting solvent. As seen in Table 1, the extraction
yield (grammes dry extract/100 g dried leaves powder) of P.
betle leaf was higher with soxhlet extraction (20.5 %)
Table 1 Extraction yield, total phenolic (TPC), total flavonoid (TFC)
and total tannin content (TTC) of P. betel leaf and grape seed extract
Extraction yielda TPCb TFCc TTCc
PBSx 20.5 203.02.7b 38.50.2b 43.52.2a
PBSn 16.2 392.96.7d 83.02.1d 68.23.7c
PBMn 18.2 281.42.8c 54.60.3c 63.01.2b
GRSx 101.71.4a 26.50.4a 61.30.4b
Except the extraction yield all other values were expressed as the mean
standard deviation (n03). Different lower case letters in the same
column were significantly different (P<0.05) from each other
a
Gramme dry extract/100 g dried leaves powder
b
Milligrammes GAE/grammes dry extract
c
Milligrammes CAE/grammes dry extract
followed by maceration (18.2 %) and sonication (16.2 %).
The observed variation in the extraction yield also reflects
the influence of the extraction techniques employed. The
increase in the extraction yield of Soxhlet extraction and
maceration might be due to the use of high extraction
temperature and longer duration of extraction, respectively.
The epidemiological studies indicated that regular con-
sumption of fruit, vegetables and herbs rich in natural antiox-
idants such as phenolics and flavonoids is associated with
reduced risk of oxidative damages due to their innate redox
properties which make them act like a reducing agents, singlet
and triplet oxygen quenchers as well as hydrogen donators
(Pietta 2000). The total phenolic (TPC) and total flavonoids
(TFC) values for P. betle leaf extracts and commercial grape
seed extract ranged between 101.71.4 to 392.96.7 mg
GAE/g dry extract and 26.50.4 to 83.02.1 mg CAE/g dry
extract, respectively. As indicated in Table 1, TPC and TFC
values were in the decreasing order of PBSn>PBMn>PBSx>
GRSx. Meanwhile, total tannins content (TTC) values ranged
from 43.52.2 to 68.23.7 mg CAE/g dry extract and the
values were in the decreasing order of PBSn>PBMn>GRSx>
PBSx. The observed significant variation in the polyphenolic
contents in the P. betle extracts might be due to the variation in
the temperature applied, duration of extraction and the extrac-
tion techniques. The presence of significant amount of poly-
phenolic content in PBSn might be due to the ability of
sonication to extract and retain most of the bioactive com-
pounds. Whereas, the decrease in the polyphenolic content in
PBSx could be due to the deterioration of some amount of
bioactive compounds at high extraction temperature. Similar
inference was also drawn by Pin et al. (2010) and Nalina and
Rahim (2007) who reported the absence of eugenol in the
extract obtained at the higher extraction temperature (100 C).
Effect of Extraction Techniques on DPPH and ABTS
Radical Scavenging Activity
Antioxidant capacity of most of the sample was evaluated
through their ability to scavenge the two commonly used
synthetic radicals such as DPPH and ABTS radicals. As
seen in Fig 1a, b, all P. betle samples and standards
exhibited the concentration-dependent percentage inhibition
of DPPH and ABTS radicals. In both the assays, the per-
centage scavenging ability of samples was in the order of
Vit. C>PBSn>PBMn>GRSx>BHT>PBSx. In DPPH as-
say, except Vit. C and PBSn none of the samples achieved
plateau at the highest concentration tested (40 g/mL).
However in ABTS assay, Vit. C achieved plateau at
10 g/mL, PBSn and PBMn at 20 g/mL whereas even at
the higher concentration tested (20 g/mL) BHT, GRSx and
PBSx did not achieved the plateau (Fig. 1b). Moreover,
strong correlation was also found between DPPH assay
and TPC (R2 00.9430) as well as TFC (R2 00.9156) and with
 Food Anal. Methods

Fig. 1 a DPPH and b ABTS

radical scavenging assay of
various extracts of P. betle and
standards; c FRAP and TAC
values, bar diagram with
different lower case letters
represents the values were
significantly different from
each other for each assay
Food Anal. Methods
TTC (R2 00.8887) which reveals the influence of polypheno-
lic content on the antioxidant activity. Furthermore, substan-
tially strong correlation was also observed between results of
ABTS assay and TPC (R2 00.9295) as well as TFC (R2 0
0.9334) and moderate correlation with TTC (R2 00.6242)
which confirms that polyphenols are likely to contribute
to the radical scavenging activity of these extracts. The
obtained significant antioxidant activity with PBSn
might be due to the presence of higher amount of
APC, eugenol, eugenyl acetate and other phenylpropa-
noids (Table 4 and 5). Rice-Evans et al. (1995) reported
the influence of number and position of hydroxyl
groups present in phenolic compounds on antioxidant
activity. Similar inferences were also drawn from our
earlier study (Annegowda et al. 2012) where in the high
phenolic and flavonoid content contributed for high
antioxidant activity through the scavenging of free
radicals.
Effect of Extraction Techniques on TAC and FRAP values
The reducing ability of antioxidants present in the samples
and standards were evaluated by TAC and FRAP assays
described by Prieto et al. (1999) and Benzie and Strain
(1996), respectively. As revealed from the Fig. 1c, all the
tested samples exhibited varying level of reducing ability
which is expressed in terms of milligrammes of VCEAC
values. In TAC assay PBSn demonstrated significant anti-
oxidant activity (P<0.05) followed by PBMn, BHT, PBSx
and GRSx with the antioxidant value of 348.31.1, 266.5
6.6, 223.55.3, 173.75.1 and 81.51.2 mg VCEAC/g dry
sample, respectively. Moreover, a strong correlation was
observed between TAC values of different sample with
TPC (R2 00.9763) TFC (R2 00.9818) and moderate correla-
tion with TTC (R2 00.6653). Similar trend of TAC was also
observed for FRAP with BPSn exhibiting significant anti-
oxidant activity (p<0.05) followed by PBMn, BHT, PBSx
and GRSx with the antioxidant value of 663.55.8, 444.9
5.8, 404.28.8, 334.21.6 and 173.92.7 mg VCEAC/g
dry sample, respectively. Moreover, the obtained antioxi-
dant value of PBSn was 3.8 and 1.6 times higher than GRSx
and BHT, respectively. In addition, strong correlation was
observed between the FRAP values and TPC (R2 00.9935),
TFC (R2 00.9955) whereas, moderate correlation with TTC
(R2 00.7376).
Antibacterial Activity
The zone of inhibition and minimum inhibitory concen-
tration of P. betle extracts and standards tested by disc
diffusion and broth dilution methods are highlighted in
Table 2. Extracts obtained from all extraction methods
demonstrated good antimicrobial activity. Among the
samples tested, PBSn exhibited higher antimicrobial ac-
tivity followed by PBMn and PBSx in disc diffusion
method. As seen in Table 2, PBSn exhibited potent
antibacterial activity against E. coli, moderate activity
against B. subtilis, S. aureus, Shigella, P. aeruginosa
and mild activity against B. cereus and K. pneumoniae
bacterial strains.
Broth dilution method described by Elof (1998) is very
simple, fast and allow multiple samples analysis at a time.
Since the samples are in direct contact with the bacterial
strains, results obtained will be accurate and reproducible.
As seen in Table 2, all the tested microbes are sensitive to
the samples as well as standards tested and the MIC values
were ranged between 0.625 and 5.0 mg/mL. Among the
samples tested, PBSn possessed highest antimicrobial activ-
ity against all tested microbes (0.625 to 1.25 mg/mL) fol-
lowed by PBMn (1.25 to 5.0 mg/mL) and PBSx (2.5 to
5.0 mg/mL). Among the microbes tested B. subtilis, P.
aeruginosa and E. coli are found to be highly sensitive
bacterial strains (MIC 0.625 mg/mL) to PBSn followed by
B. cereus, S. aureus and Shigella (MIC 1.25 mg/mL) whereas,
K. pneumoniae was found be less sensitive (MIC 2.5 mg/mL).
Though, all the samples exhibited good antimicrobial activity
in both methods, the observed activity was lesser than the
positive controls used (Table 2). Whereas, negative control
(methanol and DMSO for ZOI and MIC, respectively) did not
show any antibacterial activity at the concentration used in
both these assay models. In accordance with our findings,
Rodriguez et al. (2007) also demonstrated the influence of
phenolic compounds on antimicrobial activity against the
similar bacterial strains. Furthermore, results of our study
suggested the exploitation of P. betle leaf extract as a natural
preservative to increase the shelf life of herbal formulations as
well as nutraceuticals.
Bioautography-Guided Separation and Identification
of Antioxidants
TLC condition was optimised to obtain precise, compact
and well resolved bands from the P. betle leaf extracts and
was also used for the TLCbioautography-guided separation
of antioxidants. Of the various mobile phases tried, the
optimised mobile phase of toluene: ethyl acetate: formic
acid (3:3:0.2, v/v) gave the best resolution of eugenol (Rf 0
0.81), allylpyrocatechol (Rf 00.67) and eugenyl acetate (Rf 0
0.44) in the presence of other compound in the samples.
Preparative TLC followed bioautography lead to the sepa-
ration of three compounds (SCMP1, 2 and 3). The separated
compounds as well as corresponding well-resolved bands
from the P. betel extract were UV active, appeared as red
violet and green and yellow spots when sprayed with sul-
phuric acid, ferric chloride and DPPH (2.54 mM, methanol)
reagents, respectively.
 Food Anal. Methods

Table 2 ZOI and MIC values of

P. betel leaf extracts and stand- Name of bacteria PBSx PBSn PBMn CLMPL
ards (n03)
ZOI MIC ZOI MIC ZOI MIC ZOI MIC

B. cereus 9.5 2.5 14.0 1.25 12.5 1.25 26.5 0.2

B. subtilis 10.5 2.5 15.0 0.625 13.5 1.25 28 0.025
S. aureus 9.5 2.5 15.0 1.25 13.0 2.50 20.5 0.1
P. aeruginosa 11.0 2.5 15.0 0.625 13.5 1.25 28.5 0.025
K. pneumoniae 9.0 5.0 14.0 2.5 12.5 5.0 25.5 0.2
ZOI Zone of inhibition, MIC E. coli 11.0 2.5 17.0 0.625 14.5 1.25 25 0.025
Minimum inhibitory concentra- Shigella 10.0 2.5 15.0 1.25 12.0 1.25 26 0.05
tion, CLMPL chloramphenicol

HPTLC Method Validation and Quantification of Identified
Antioxidants
Recently, several researchers reported the very close simi-
larity in the HPLC and HPTLC chemical fingerprinting for
several plants (Gallo et al. 2008; Pobocka-Olech et al.
2010). Hence, we employed HPTLC method to obtain the
chemical fingerprint of P. betle leaf as well as to quantify the
phytoconstituents resolved by TLC and identify the separat-
ed compounds. The HPTLC densitogram (Fig. 2a) repre-
sents the qualitative analysis of various extracts of P. betle
leaf using HPTLC coupled with DAD detector at 280 nm.
As seen in Fig. 2a, all the resolved bands were varied
qualitatively as well as quantitatively, out of seven resolved
bands, band 2, 3, 5 and 6 were antioxidants in nature as they
shown yellow colour after spraying with DPPH reagent.
Separated compound 1, 2 and 3 (SCMP1, 2 and 3) were
identified as eugenol, allylpyrocatechol and eugenyl acetate
by the overlay of HPTLC chromatogram, by comparing the
Rf values, UV spectra and colour displayed by them with the
spraying reagents to the authentic standards analysed under
similar HPTLC condition. Purity of the eugenol, APC and
eugenyl acetate as well as the corresponding well re-
solved bands from extract was confirmed as the absorp-
tion spectra at the start, middle and end position of the
bands were comparable.
The HPTLC method developed for the simultaneous
analysis of eugenol, APC and eugenyl acetate was validated
in terms of precision, repeatability, specificity and accuracy
(Table 3). The linearity range for eugenol and APC was
found to be 2501,500 ng whereas, for eugenyl acetate it
was 5003,000 ng. The regression equation and correlation
coefficient for eugenol, APC and eugenyl acetate was y0
9.5426x +719.66 (R2 00.9990), y01.3061x83.779 (R2 0
0.9990) and y00.5415x9.856 (R2 00.9994), respectively
(Table 3). This revealed good linearity response for the
developed HPTLC method. As seen in the Table 3, the
LOD and LOQ were found to be 40 and 250 ng for eugenol,
60 and 250 ng for APC and 100 and 500 ng for eugenyl
acetate, respectively. The intra-day and inter-day precision
and repeatability (Table 3) expressed in terms of percentage
coefficient of variance (%CV) demonstrated the good pre-
cision and reproducibility of this method. It is also evident
from Table 3, the percentage recovery of eugenol, APC and
eugenyl acetate was 99.42, 99.15 and 99.31, respectively,
which indicates that developed method is accurate and
reliable.
The amount of eugenol, APC and eugenyl acetate
determined by HPTLC method in the various extracts
of P. betle leaf sample was ranged between 57.03
78.55, 91.09101.52 and 55.0470.55 mg/g samples
respectively (Table 4). This result also indicates the
influence of the extraction techniques on the extraction
of potent bioactive compounds with PBSn containing
higher amount of them followed by PBMn and PBSx,
and also supported the in vitro antioxidant activity and
polyphenolic constituents evaluated.
GCMS Analysis of Phytoconstituents in Various Extracts
of P. betle Leaf
Plants synthesise and store wide range of phytoconstituents
with varying amount which necessitates the utilisation of
several analytical techniques for their evaluation. P. betle is
one of the therapeutically important plants containing nu-
merous volatile components which may not ionise optimally
by LCMS along with few non volatile constituents. Hence,
we used GCMS method for the analysis of various phyto-
constituents. Fig. 2b represents the GCMS of P. betle
extracts obtained from various extraction techniques and
Table 5 represents the retention time (Rt), molecular mass
and the amount of resolved phytoconstituents in terms of
peak area. The region (Rt between 7.5 and 12) in which most
of the antioxidants well resolved were only displayed in the
Fig. 2b. Resolved compounds were identified by comparing
the fragmentation patterns of them with those published in
literature and using NIST database of GCMS (more than
90 % probable) and included in Table. 5. The phytocon-
stituents with therapeutical and nutraceuticals importance
such as eugenol, eugenyl acetate, APC, APC monoacetate,
Food Anal. Methods

Fig. 2 a HPTLC densitogram

of different extracts, isolated
a
compounds and biomarkers of
P. betle at 280 nm; b GCMS
chromatogram of P. betel leaf
extracts

b
Abundance

TIC: PB sonication H.D

4500000 8.26

4000000

3500000

3000000

9.29
2500000
7.57
11.63

2000000
8.58
8.49 11.68
1500000

1000000 11.94
11.82
7.74 10.74
7.65 7.96
500000 8.03
10.92
8.42
11.08

7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50

Time-->

Table 3 HPTLC validation

parameters for eugenol, allyl- Parameters Eugenol Allylpyrocatechol Eugenyl acetate
pyrocatechol and eugenyl
acetate Instrumental precision (%CV) 0.89 0.86 1.37
Repeatability (%CV) 0.52 1.55 1.07
Accuracy (% recovery) 99.42 99.15 99.31
Limit of detection (ng) 40 60 100
Limit of quantification (ng) 250 250 500
Specificity Specific Specific Specific
Linearity (correlation coefficient) 0.9990 0.9990 0.9994
Range (ng/band) 2501,500 2501,500 5003,000
Intra-day precision 1.47 1.35 1.08
Inter-day precision 1.02 1.56 1.47
 Food Anal. Methods

Table 4 Marker compounds quantified by HPTLC method from var- Conclusions

ious samples of P. betle leaf

Samples Content of marker compoundsa (mg/g dried sample) This is the first ever study conducted regarding the TLC
bioautography-guided separation; HPTLC as well as GC
Eugenol Allylpyrocatechol Eugenyl acetate MS-assisted identification and quantification of antioxidants
from the various ethanolic extracts of P. betle leaf. The
PBSx 57.031.23 91.091.07 55.040.76
results of present study confirm the influence of extraction
PBSn 78.551.67 101.521.75 70.552.26
techniques on polyphenolic content, antioxidant and antimi-
PBMn 69.810.57 99.741.03 63.060.95
crobial activities. Though, Soxhlet, sonicated and macerated
a
Mean valuesSD (n03) extracts of P. betle leaf exhibited potent antioxidant and
antimicrobial activity, the found activity was varied signif-
icantly depending on the method of extraction employed.
The GCMS analysis as well as validated HPTLC method
APC diacetate, phytol, myristic acid and -sitosterol also revealed the presence of various phytoconstituents in
found from GCMS study were present in varying varying amount in the extracts obtained by different extrac-
amount in the extracts obtained from various extraction tion techniques which also affirm the in vitro results. More-
techniques. Most of these constituents were present in over, most of the herbal formulations containing P. betel as
higher amount in PBSn followed by PBMn and PBSx one of the ingredient are exhibiting their claimed beneficial
which however contain saturated fatty acids in higher effects through antioxidant mechanism. Hence, results of
amount. Results of GCMS analysis also explain the this study also seize the attention of the herbal industry to
reason for the significant antioxidant, antimicrobial ac- select the suitable extraction technique to obtain antioxidant
tivities and polyphenolics contained in PBSn in compar- rich P. betel leaf extract prior to include in the herbal
ison with PBMn and PBSx and is concurrent with the formulations. Results of this study also provide evidence
results of HPTLC study. linking the antioxidant efficacy of P. betel leaf extract to the

Table 5 Phytochemical compo-

sition of various extracts of P. Phyotcompounds Rt (min) Molecular mass Peak area
betel leaf by GCMS
PBSx PBSn PBMn

1,8 Cineole 4.86 154 38,148 47,613 41,902

-Myrcene 5.44 136 11,124 14,128 16,808
-Elemecene 7.17 136 204,165 180,608 151,750
Eugenol 7.57 164 962,357 2,165,663 1,937,486
-Cubene 7.66 204 286,757 520,812 452,322
-Pinene 7.75 204 505,902 573,401 107,379
-Caryophyllene 7.98 204 133,856 542,365 461,535
Copaene 8.03 204 8,842 338,881 284,976
Allylpyrocatechol (APC) 8.27 150 4,347,207 4,635,605 4,520,406
-Cardinene 8.43 204 191,538 298,386 258,042
APC monoacetate 8.49 192 809,431 1,447,151 1,370,005
Eugenyl acetate 8.59 206 1,175,233 1,847,317 1,540,359
APC diacetate 9.29 234 3,379,736 2,541,358 1,750,720
Tridecanoic acid methyl ester 10.75 270 626,640 547,991 304,963
Palmitic acid 10.93 256 688,398 371,644 237,988
Myristic acid 11.08 228 249,693 182,052 53,323
Stearic acid 11.63 284 2,230,287 2,000,564 831,433
Phytol 11.68 296 1,453,641 281,460 1,132,757
Octadeca trienoic acid ethyl ester 11.83 306 1,091,742 860,505 614,675
Campesterol 17.51 400 59,192 99,568 82,516
Rt retention time, GCMS gas Stigmasterol 17.79 412 44,938 78,843 59,672
chromatography mass spectrom- -Sitosterol 18.34 414 260,609 408,814 349,022
etry, APC allylpyrocatechol
Food Anal. Methods
developed HPTLC fingerprint. The developed HPTLC method
for the simultaneous evaluation of eugenol, APC and eugenyl
acetate was found to be very simple, precise, specific, sensitive
and accurate and can be utilised for the routine quality control
of P. betle leaf extracts or the herbal formulation containing it.
Further study is in progress for the separation and identification
of remaining bioactives from this plant and their simultaneous
estimation using HPLC and HPTLC methods.
Acknowledgments This project was funded by USM Research
University Grant, Malaysia. The author (Annegowda H.V.) grate-
fully acknowledges the Institute of Postgraduate Studies of USM,
Malaysia for granting USM Fellowship.
References
Abou-Donia AH, Toaima SM, Hammoda HM, Shawky E (2008) New
rapid validated HPTLC method for the determination of
galanthamine in Amarallidaceae plant extracts. Phytochem
Anal 19:353358
Annegowda HV, Mordi MN, Ramanathan S, Hamdan MR, Mansor SM
(2012) Effect of extraction techniques on phenolic content, anti-
oxidant and antimicrobial activity of Bauhinia purpurea: HPTLC
determination of antioxidants. Food Anal Methods 5:226223
Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD (2005a)
Anti-diabetic activities of aqueous and ethanolic extracts of Piper
betle leaves in rats. J Ethnopharmacol 102:239245
Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD (2005b)
Antinociceptive activities of aqueous and ethanol extracts of
Piper betle leaves in rats. Pharm Biol 43(9):766772
Bauer AW, Kirby WMM, Sherris JC, Truck M (1966) Antibiotic
susceptibility testing by a standardised single disc method. Am J
Clin Pathol 45:493496
Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma
(FRAP) as a measure of antioxidant power, the FRAP assay. Anal
Biochem 239:7076
Blois MS (1958) Antioxidant determinations by the use of a stable free
radical. Nature 181:11991200
Choudhary D, Kale RK (2002) Antioxidant and non-toxic properties of
Piper betle leaf extract: in vitro and in vivo studies. Phytother Res
16:461466
Elof JN (1998) A sensitive and quick microplate method to determine
the minimal inhibitory concentration of plant extracts for bacteria.
Planta Medica 64:711713
Gallo FR, Multari G, Giambenedetti M, Federici E (2008) Chemical
fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and
densitometry. Phytochem Anal 19:550559
Gu L, Wu T, Wang Z (2009) TLC bioautography-guided isolation of
antioxidants from fruit of Perilla frutescens var. acuta. LWT
Food Sci Technol 42:131136
Jantan I, Ahmad AR, Ahmad AS, Ali AM (1994) A comparative study
of the essential oils of five Piper species from Peninsular Malaysia.
Flavour Frag J 9:339342
Jeng JH, Chen SY, Liao CH, Tung YY, Lin BR, Hahn LJ, Chang MH
(2002) Modulation of platelet aggregation by areca nut and betle
leaf ingredients: roles of reactive oxygen species and cyclooxy-
genase. Free Radic Biol Medicine 32:860871
Majumdar B, Chaudhuri SR, Ray Arun, Bandyopadhyay SK (2002)
Potent antiulcerogenic activity of ethanol extract of leaf of Piper
betle linn by antioxidative mechanism. Indian J Clin Biochem
17:4957
Nalina T, Rahim ZHA (2007) The crude aqueous extract of Piper betle
L. and its antibacterial effect towards Streptococcus mutans. Am J
Biotechnol Biochem 3:1015
Nantitanon W, Yotsawimonwat S, Okonogi S (2010) Factors influencing
antioxidant activities and total phenolic content of guava leaf extract.
LWTFood Sci Technol 43:10951103
Pietta P (2000) Flavonoids as antioxidants. J Nat Products 63:1035
1042
Pin KY, Chuah AL, Rashih AA, Mazura MP, Fadzureena J, Vimala S,
Rasadah MA (2010) Antioxidant and anti-inflammatory activities
of extracts of betel leaves (Piper betle) from solvents with different
polarities. J Trop For Sci 22:448455
Pobocka-Olech L, Krauze-Baranowska M, Gd D, Kawiak A, ojkowska
E (2010) Chromatographic analysis of simple phenols in some species
from the genus Salix. Phytochem Anal 21:463469
Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric quantitation
of antioxidant capacity through the formation of phosphomolybdenum
complex: specific application to the determination of vitamin E. Anal
Biochem 269:337341
Rajani M, Ravishankara MN, Shrivastava N, Padh H (2001) HPTLC-
aided phytochemical fingerprinting analysis as a tool for evaluation
of herbal drugs. A case study of Ushaq (Ammoniacum gum). J
Planar Chromatogr 14:3441
Ramji N, Iyer R, Chandrasekaran S (2002) Phenolic antibacterials from
Piper betle in the prevention of halitosis. J Ethnopharmacol
83:149152
Rathee JS, Patro BS, Mula S, Gamre S, Chattopadhyay S (2006)
Antioxidant activity of Piper betle leaf extract and its constituents.
J Agric Food Chem 54:90469054
Rawat AKS, Tripathi RD, Khan AJ, Balasubrahmanyam VR (1989)
Essential oil components as markers for identification of Piper
betle L. cultivars. Biochem Syst Ecol 17:3538
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C
(1999) Antioxidant activity applying an improved ABTS radical
cation decolorizing assay. Free Radic Biol Medicine 26:1231
1237
Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB
(1995) The relative antioxidant activities of plant-derived
polyphenolic flavonoids. Free Radic Res 22:375383
Rodriguez VMJ, Alberto MR, Manca de Nadra MC (2007) Antibacterial
effect of phenolic compounds from different wines. Food Control
18:93101
Sakanaka S, Tachibana Y, Okada Y (2005) Preparation and antioxidant
of extracts of Japanese persimmon leaf tea (kakinoha-cha). Food
Chem 89:569575
Santhanam G, Nagarjan S (1990) Wound healing activity of Curcuma
aromatica and Piper betle. Fitoterapia 61:458459
Sarkar M, Gangopadhyay P, Basak B, Chakrabarty K, Banerji J,
Adhikary P, Chatterjee A (2000) The reversible antifertility effect
of Piper betle Linn. on Swiss albino male mice. Contraception
62:271274
Sharma ML, Rawat AKS, Balasubrahmanyam VR, Singh A (1983)
Studies on essential oil of betelvine leaf (Piper betle Linn.). Part
II. Indian Perfum 27:9193
Simes-Pires CA, Hmicha B, Marston A, Hostettmann K (2009) A
TLC bioautographic method for the detection of - and -
glucosidase inhibitors in plant extracts. Phytochem Anal
20:511515
Singh M, Shakya S, Soni VK, Dangi A, Kumar N, Bhattacharya SM
(2009) The n-hexane and chloroform fractions of Piper betle L.
trigger different arms of immune responses in BALB/c mice and
exhibit anti filarial activity against human lymphatic filarid
Brugia malayi. Int Immunopharmacol 9:716728
Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am J Enol
Vitic 16:144158

Sun BS, Ricardo-Da-Silva JM, Spranger MI (1998) Critical factors of
vanillin assay for catechins and proanthocyanidins. J Agric Food
Chem 46:42674274
Tappayuthpijarn P, Dejatiwongse Q, Pongpech P, Leelaporn A (1982)
Antibacterial activity of extracts of Piper betle leaf. Thai J Phar-
macol 4:205212
Wang L, Weller CL (2006) Recent advances in the extraction of
nutraceuticals from plant. Trends Food Sci Tech 17:300312
Wirotesangthong M, Inagaki N, Tanaka H, Thanakijcharoenpath W,
Nagai H (2008) Inhibitory effects of Piper betle on production of
Food Anal. Methods
allergic mediators by bone marrow-derived mast cells and lung
epithelial cells. Int Immunopharmacol 8:453457
Young SC, Wang CJ, Lin JJ, Peng PL, Hsu JL, Chou FP (2007)
Protection effect of Piper betle leaf extract against carbon
tetrachloride-induced liver fibrosis in rats. Arch Toxicol
81:4555
Zeng HW, Jiang YY, Cai DG, Bian J, Long K, Chen ZL (1997)
Piperbetol, methylpiperbetol, piperol A and piperol B: a new
series of highly specific PAF receptor antagonists from Piper
betle. Planta Medica 63:296298