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Isolation of Allylpyrocatechol from Piper betle

L. Leaves by Using High-Performance Liquid
Chromatography
a a
Noor Faradilla Abdullah & Roslinah Mohamad Hussain
a
Department of Medical Laboratory Technology, Faculty of Health Sciences , Universiti
Teknologi MARA (UiTM) , Selangor , Malaysia
Accepted author version posted online: 20 May 2014.Published online: 10 Oct 2014.

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To cite this article: Noor Faradilla Abdullah & Roslinah Mohamad Hussain (2015) Isolation of Allylpyrocatechol from Piper
betle L. Leaves by Using High-Performance Liquid Chromatography, Journal of Liquid Chromatography & Related Technologies,
38:2, 289-293, DOI: 10.1080/10826076.2014.908782

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 Journal of Liquid Chromatography & Related Technologies, 38: 289–293, 2015
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2014.908782

Isolation of Allylpyrocatechol from Piper betle L. Leaves by

Using High-Performance Liquid Chromatography
NOOR FARADILLA ABDULLAH and ROSLINAH MOHAMAD HUSSAIN
Department of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA (UiTM),
Selangor, Malaysia

Piper betle L. plant or ‘‘Sirih’’ is widely found in Southeast Asia and its leaves are commonly eaten together with lime and arecanut.
Downloaded by [University of Otago] at 06:23 24 December 2014

The major active compounds of P. betle leaves are polyphenols, for example, allylpyrocatechol (APC), that are reported to possess
anti-inflammatory, antioxidant, and antimicrobial properties. We developed and validated a method for quantitative determination
of APC in the extracts from betel leaves using high-performance liquid chromatography (HPLC). Extraction of APC was
performed using Agilent 1100 series HPLC on a ZORBAX Eclipse column (C18 with 250 mm
4.6 mm) with acetonitrile—
0.01 M phosphoric acid as the mobile phase under gradient mode. The flow rate was 1.0 mL=min, and APC was detected using
a UV detector (222 nm). Concentration of APC in the extract was determined from the peak areas by referencing them against
a calibration curve developed from commercial analytical APC standard. The yield of APC from this HPLC extraction was
13.82% compared to the isolation of APC using silica gel column chromatography in which the yield of APC eluted was only
0.9% (w=w) of the extract (4.7 g). APC fraction isolated from this study will be used for the molecular analysis of its effect on
regulation of oxidative stress response enzymes in Staphylococcus aureus. In addition, this study potentially helps in choosing
the suitable extraction technique and developed HPLC method that can be used for routine quality control and standardized
formulations of APC in P. betle leaf extract.
Keywords: allylpyrocatechol extraction, analytical HPLC, quantitative determination, yield, Piper betle L., ethanolic extract

Introduction constituent showed antimicrobial properties against

Piper betle L. plant belongs to the genus Piper of Piperaceae
family, and betel leaves are aromatic creepers that possess
smooth, shining, alternate, and long stalked leaves with
pointed apex.[1] It is widely used among the Indians and
Malays in Malaysia for recreational use. The pungent and
aromatic flavors of the leaves are widely consumed as
a mouth freshener.[2] Its promising traditional uses have
led to many biological and chemical studies, and many
scientific studies have reported the biological properties of
P. betle L. including anti-inflammatory, antioxidant, and
antimicrobial properties.[3–5] Extract of P. betle L. leaves
are known to possess high antioxidant properties and exert
antiproliferative effects against breast cancer (MCF-7) cell
line.[6] Further, ethanolic extract of P. betle L. leaves has
been shown to exert a significant antibacterial effect on
Staphylococcus aureus and effectively reduce catalase
activity in this organism.[7] The leaf extract and its phenolic
Address correspondence to: Roslinah Mohamad Hussain,
Department of Medical Laboratory Technology, Faculty of
Health Sciences, Universiti Teknologi MARA (UiTM), Puncak
Alam Campus, Level 17, FSK6, 42300 Puncak Alam, Selangor,
Malaysia. E-mail: roslinah561@salam.uitm.edu.my
Color versions of one or more of the figures in the article
can be found online at www.tandfonline.com/ljlc.
a number of oral bacteria, including Streptococcus mutans.[8]
The important phenolic constituents isolated from the leaves
of P. betle L. include eugenol, eugenol acetate, allylpyrocate-
chol (APC), allylpyrocatechol monoacetate, and chavibetol
acetate.[9] APC has been shown to exert anti-inflammatory
effect in a mouse model of inflammation[10] and protective
effect against indomethacin-induced gastric ulceration in the
mouse model.[2]
The most essential steps in the isolation of target
compound from plant extracts are extraction and product
recovery. The nature and amount of bioactive compounds
isolated from the plant material mainly depend on the type
of extraction technique, temperature, polarity of polar solvent
used, and duration of extraction.[11] Previous method of APC
isolation was performed using silica-gel chromatography,
which requires a combination of solvents in extraction
process.[10] Owing to the current interest in the potential
effects of therapeutic agent from natural products, this study
attempted to isolate the major active compound, APC,
using rapid high-performance liquid chromatography
(HPLC) technique. The chemical structure of APC is shown
in Figure 1.
Allylpyrocatechol is a polar compound with empirical for-
mula of C9H10O2(MW 150.17). It is a phenolic compound
consisting of two hydroxyl group (–OH) located at C3 and
C4 of benzene ring, which gives it a more polar characteristic.
 290
Fig. 1. Chemical structure of allylpyrocatechol (APC).
Other synonyms are 4-allylpyrocatechol, 1,2-dihydroxy-4-
allylbenzene, 4-allylcatechol, 4-allyl-1,2-benzenediol, and
4-(2-propenyl)-1,2-benzenediol.[12]
The objective of this study was to develop a more efficient
and less time-consuming method for the isolation of APC
from betel leaf extract. To the best of our knowledge, this is
the first report on the isolation of APC by using HPLC analy-
sis. In this study, betel leaf extraction was conducted using
95% ethanol as solvent. The extract was analyzed using ana-
lytical HPLC to detect and quantify APC from P. betle leaves.
Downloaded by [University of Otago] at 06:23 24 December 2014
The percentage of APC yield was 13.82%. The optimized
method for recovery of APC, which was developed in this
study, will be applied to preparative HPLC to develop a
method for high-throughput purification of APC. The effects
of APC on the oxidative stress response enzymes in S. aureus
will be investigated in our further downstream research.
Experimental
Plant Extraction
Piper betle L. leaves were collected from Ijok, Selangor, and
authenticated at the Forest Research Institute Malaysia (PID
541213-31). The leaves were washed with water and dried
in an oven (Labtech LCO-3050H, Daihan Labtech Co., Ltd,
Korea) at 40 C. Dried leaves were ground using a mechanical
blender (Khind BL-1012, Khind-Mistral (M) Sdn Bhd,
Malaysia) into powdered form and stored at 20 C until
use. For preparation of extract, powdered leaves were mixed
with 95% ethanol (1 g: 20 mL) and incubated at room tempera-
ture for 24 hr. The mixture was filtered to remove debris and
evaporated using a rotary evaporator (Eyela OSB-2100). The
dried extract was collected and stored at 4 C before analysis.[13]
Reagents
Acetonitrile and phosphoric acids used in the study were of
liquid chromatography grade (Merck, Whitehouse Station,
NJ, USA). Analytical standard of APC was obtained from
Sigma-Aldrich (M) Sdn Bhd (Selangor, Malaysia) and working
standards for the calibration curve, ranging in concentration
from 0.01 to 0.2 mg=mL, were prepared by dilution of stock
with acetonitrile. All standard solutions were stored in dark
at 4 C. Water used in this study was obtained from Millipore.
HPLC Analysis
The dried extract from 95% ethanol (1.0 mg) was dissolved in
1.0 mL acetonitrile (CH3CN) and filtered (17 mm, porosity:
0.45 mm, poly(vinylidene difluoride) membrane) before
HPLC analysis. We injected 5 mL (0.005 mg) extract into
HPLC for analysis to determine the chemical profile and con-
centration of APC. HPLC analysis was carried out using Agi-
lent 1100 series HPLC with a ZORBAX Eclipse column (C18
N. F. Abdullah and R. M. Hussain
Table 1. The change of mobile phase in HPLC analysis
100% Acetonitrile 0.1% Phosphoric
Time (min) (%) acid (%)
0.0 5 95
1.0 5 95
11.0 50 50
15.0 50 50
with 250 mm
4.6 mm, 5 mm particle size) as stationary
phase. Column conditioning was conducted by flushing it with
10–20 column volumes of solution consisting of 90% acetoni-
trile and 10% water. Extraction was conducted at a flow rate
of 1.0 mL=min and detected at 222 nm with a UV detector,
as described in the HPLC QC Method for APC standard
(Sigma-Aldrich).[12] The mobile phase was in gradient mode
and consisted of 100% acetonitrile and 0.1% phosphoric acid
in water, with varying gradient ratios, as shown in Table 1.
A quantitative determination of APC in the extract was
performed by comparing the peak area from the sample
injection to the corresponding area from standard injection
on the calibration curve. The amount of APC present
in a sample was measured using the standard calibration
curve. All analyses were run in triplicate.
The calculation of yield was based on calibration curve,
given by:[14,15]
AI
Concentration of the compound :
M
where A, the peak area of the compound in test solution; I,
the y-intercept of the calibration curve; and M, the slope of
the calibration curve.
The percentage of APC in the sample can be calculated
using the following equation:[14,15]
C
V
D
Percentage of APC :
10; 000 W
Fig. 2. Calibration curve of APC standard. Working APC
standards ranged from 1 (0.01 mg=mL), 2 (0.05 mg=mL), 3
(0.1 mg=mL) and 4 (0.2 mg=mL). The concentration of extract
was calculated from the calibration curve (138.218 ng=mL).
 Isolation of Allylpyrocatechol from Piper betle L. Leaves 291
Table 2. Linearity parameters for the calibration curve of APC Table 3. Retention time and area of APC peak in P. betle L.
standard using HPLC analysis ethanolic extract obtained from HPLC chromatogram
Slope Intercept r2 Retention time Area (mAU)

28.07004 117.42982 0.995 13.734 3997.20410

Working range: 0.01–0.2 mg=mL, r2: Correlation coefficient.

standard (13.777 min), as shown in Figure 4. From the

where C, the concentration (in mg=L) of the analyte in
the test solution; V, dilution factor, if any; D, the final
make up volume (in mL) of the test solution; and W,
the weight (in g) of the sample used for the preparation
of the test solution.
optimized HPLC extraction method developed in this study,
APC was found to be the major chemical compound found
in P. betle L. ethanolic extract.
Calculation of APC Yield

Results and Discussion The calculation of yield was based on calibration curve,
given by:
Response Linearity
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3997:20410  117:42982
APC standard was prepared in four concentrations: 0.01, Concentration of the compound :
28:07004
0.05, 0.1, and 0.2 mg=mL. The peaks of APC standards were : 138:21 mg=L
integrated and identified, and the peak areas were plotted
against concentration to give a calibration curve (Figure 2).
The correlation coefficient from the regression analysis was Calculation of Percentage of APC Yield
determined to be 0.995, which was within the acceptance
138:21
1
limits (Table 2, acceptance criteria (r2) > 0.98). Percentage of APC :
10000ð0:001 gÞ
: 13:82%
Extraction Yield
HPLC chromatogram of P. betle L. extract showed a major
peak corresponding to APC with retention time at 13.734 min From the calculation, the APC concentration in the extract
(Figure 3). The retention time and peak area are shown in was determined to be 138.21 ng=mL, showing a percentage
Table 3. The important parameters, including retention time yield of 13.82%. Results of this work indicate that APC
and wavelength with maximum absorbance (222 nm), of the readily extracted from P. betle leaves by the optimized
APC peak in extract were the same as those of the APC HPLC method appeared as the major component of the

Fig. 3. HPLC chromatograms of P. betle L. ethanolic extract showing one major peak with retention time at 13.734 min. Reversed
phase separation was performed using a C18 Zorbax Eclipse column (250 mm
4.6 mm). The mobile phase consisted of acetonitrile
(Solvent A) and phosphoric acid in water (Solvent B). The flow rate was 1.0 mL=min and the gradient programme consisted of:
5–95% B for 10 min and 50–50% B for 5 min. The eluted peaks were monitored at 222 nm. 5 mL of sample was injected into the
HPLC.
 292
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Fig. 4. HPLC chromatograms of (a) standard allylpyrocatechol (APC) with concentration of 0.2 mg=mL (shown at 13.777 min) and
(b) sample of extract with retention time at 13.734 min detected at 222 nm.
betel leaf extract. In comparison, the isolation of APC using
column chromatography reported in another study showed
an APC yield of only 0.9% (w=w) of the extract in elution
fraction (4.7 g).[10]
Most of the previous researchers have used silica gel
column chromatography for isolation of APC.[2,10,16] In their
study, APC was eluted with combination of ethyl acetate and
hexane, which are moderately polar and nonpolar solvents,
respectively. Further, the combination of solvents used in
the column chromatography method is sometimes difficult
to remove solvents completely from the rotary evaporator.[17]
This isolation method has sample cleanup steps following the
extraction procedure, which are accomplished by solid-phase
extraction or liquid–liquid extraction before the analytical
column in the HPLC analysis.
In the initial plant extraction of this study, the leaves were
dried at 40 C in an oven to avoid degradation of active
compounds. If high temperatures are used in the extraction
method, it can lead to the degradation of heat-sensitive com-
pounds.[18] Different solvent systems are available to extract
the active compounds from natural product. The extraction
of hydrophilic compounds uses polar solvents such as etha-
nol, methanol, or ethyl acetate.[19] The presence of hydroxyl
group in ethanol as extraction solvent was advantageous in
extracting APC because its hydroxyl group could bind with
that present in APC. Furthermore, this study used higher
amount of moderately polar solvent, acetonitrile, in HPLC,
which helps decrease elution (retention) time. Gradient
elution uses this effect by automatically reducing the polarity
and the surface tension of the aqueous mobile phase during
the course of the analysis.[11] In addition, APC, which has
a higher polar surface area (–OH) and branched chain
compound, elutes more rapidly. The use of higher polar sol-
vent in HPLC resulted in shorter eluting time and higher
yield of APC. Hence, we used HPLC technique to obtain a
much simpler and efficient method for isolation of APC.
N. F. Abdullah and R. M. Hussain
This study has shown that isolation of APC can be
accelerated with optimization of the recovery and extraction
of this compound using HPLC. In comparison to silica gel
column chromatography, it is a time-consuming procedure
that relies on force of gravity during extraction. However,
the consideration for the high cost in HPLC must be taken into
account. The applied HPLC method was found to be rapid,
simple, precise, and accurate for the determination and
isolation of APC from betel leaf extract. In addition, APC
isolated in this study is then used for the determination of other
biological properties and can be easily and conveniently
applied for routine analysis of APC in quality control.
Acknowledgment
The authors acknowledge the technical assistance provided
by the Analytical Unit, Faculty of Pharmacy, UiTM, in
carrying out HPLC analysis.
Funding
The authors are thankful to the Faculty of Health Sciences,
Universiti Teknologi MARA (UiTM) and the Federal
Research Grant Scheme (FRGS 2011) from the Ministry
of Science Technology and Innovation Malaysia for the
funding provided to enable this project.
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