Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: https://www.tandfonline.com/loi/lanl20

Determination of the Volatile Compounds in Five

Varieties of Piper betle L. from Bangladesh Using
Simultaneous Distillation Extraction and Gas
Chromatography/Mass Spectrometry (SDE-GC/MS)

Md Atikul Islam, Keun Young Ryu, Naeem Khan, Ok Yeon Song, Ji Young
Jeong, Ji Hyeon Son, Nargis Jamila & Kyong Su Kim

To cite this article: Md Atikul Islam, Keun Young Ryu, Naeem Khan, Ok Yeon Song, Ji Young
Jeong, Ji Hyeon Son, Nargis Jamila & Kyong Su Kim (2020) Determination of the Volatile
Compounds in Five Varieties of Piper�betle L. from Bangladesh Using Simultaneous Distillation
Extraction and Gas Chromatography/Mass Spectrometry (SDE-GC/MS), Analytical Letters, 53:15,
2413-2430, DOI: 10.1080/00032719.2020.1744160

To link to this article: https://doi.org/10.1080/00032719.2020.1744160

Published online: 10 Apr 2020.

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ANALYTICAL LETTERS
2020, VOL. 53, NO. 15, 2413–2430
https://doi.org/10.1080/00032719.2020.1744160

CHROMATOGRAPHY

Determination of the Volatile Compounds in Five Varieties

of Piper betle L. from Bangladesh Using Simultaneous
Distillation Extraction and Gas Chromatography/Mass
Spectrometry (SDE-GC/MS)
Md Atikul Islama,b, Keun Young Ryuc, Naeem Khand, Ok Yeon Songa, Ji Young
Jeonga, Ji Hyeon Sona, Nargis Jamilae, and Kyong Su Kima
a
Department of Food and Nutrition, Chosun University, Gwangju, Republic of Korea; bDepartment of
Chemistry, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh; cHealth
and Environment Research Institute of Gwangju, Gwangju, Republic of Korea; dDepartment of
Chemistry, Kohat University of Science & Technology, Kohat, Pakistan; eDepartment of Chemistry,
Shaheed Benazir Bhutto Women University, Peshawar, Pakistan

ABSTRACT ARTICLE HISTORY

Five varieties of Piper betle L. var. leaves including Bangla, Sanchi, Received 31 January 2020
Misti, Khasia, and BARI Paan 3 from Bangladesh were subjected to Accepted 15 March 2020
the analysis of their volatile compounds. Simultaneous distillation
KEYWORDS
extraction (SDE) with 1:1 (v/v) n-pentane:diethylether was used for
Eugenol; Piper betle L;
the isolation of the volatile compounds while the analysis was done simultaneous distillation
by gas chromatography/mass spectrometry (GC/MS). From the extraction–gas chromatog-
results, the Misti betel leaf variety showed the highest concentration raphy/mass spectrometry
of volatile compounds (13959 mg/kg) followed by BARI Paan 3 (SDE-GC/MS); volatile
(11684 mg/kg), Khasia (11110 mg/kg), Sanchi (6959 mg/kg) and compounds
Bangla (4347 mg/kg). A total of 101 compounds were identified in
which 42 were present in all of the varieties but at varying concen-
trations. The present study reports 50 volatile compounds in betel
leaves for the first time in the literature. Eugenol was found in all of
the varieties at the highest concentration value followed by b-cary-
phyllene, c-muurolene, valencene, eucalyptol, chavicol and caryphel-
lene oxide. Based on the volatile compounds, the five varieties of
betel leaf were differentiated into three clusters by the use of princi-
pal component analysis (PCA). Hierarchical cluster analysis (HCA) also
divided the leaf varieties into three clusters composed of Misti and
BARI Paan 3, Bangla and Sanchi, and Khasia.

Introduction
Piper betle L. var. is a perennial creeper belonging to the family Piperaceae, genus
Piperis and its English name is betel leaf. These plants are extensively grown and used
in Bangladesh, India, Pakistan, Malaysia, Indonesia, Thailand, Sri Lanka, Madagascar,
and in the West Indies (Karak et al. 2016). These leaves have different names in various
countries around the world; however “Paan” is the most popular name in Bangladesh,
India, Pakistan, and Nepal (Guha and Nandi 2019). Betel leaves are consumed in

CONTACT Kyong Su Kim kskim@chosun.ac.kr Department of Food and Nutrition, Chosun University, Gwangju
61452, Republic of Korea.
ß 2020 Taylor & Francis Group, LLC
2414 M. A. ISLAM ET AL.

different forms by more than 600 million people around the world and is the third
most consumed worldwide after tea and coffee (Pradhan, Biswasroy, and Suri 2014).
Bangladesh is the second largest producer of betel leaves in the world (Peter 2014). It
has been reported in the literature that in Bangladesh betel leaves are cultivated on
approximately 12,660 to 18,247 hectares of farmland, with an annual production of 0.06
to 0.10 million tons (Islam et al. 2015). Some studies have confirmed that there are over
100 varieties of betel leaves around the world (Guha 2006). According to Ravindran,
Johny, and Nirmal (2002) the number of betel leaf varieties is close to 150.
The aroma and bioactivity of betel leaves is primarily dependent upon the volatile com-
pounds present in its essential oil (Das et al. 2016). The betel leaf essential oil is slightly
greasy, viscous, and slippery liquid at room temperature and has a strong pungent aro-
matic flavor. Betel leaves contain various phytochemicals such as eugenol, chavicol, methyl
eugenol, chavibetol, hydroxychavicol, hydroxycatechol, b-caryophyllene, estragole 1,8-cin-
eol, a-pinene, and b-pinene. These leaves have many applications in the food, cosmetic
and pharmaceutical industries around the world (Guha and Nandi 2019).
A wide variety of volatile active compounds have been reported in the literature from
betel leaves, which vary depending on the type of landrace, cultivation location, envir-
onment, and soil (Suryasnata et al. 2016). Volatile oil from betel leaves may be extracted
using different methods that have been applied for the isolation for essential oil-bearing
crops. These protocols include hydrodistillation (Das et al. 2016), steam distillation
(Alighiri et al. 2018), supercritical fluid extraction (Singtongratana, Vadhanasin, and
Singkhonrat 2013) and solvent extraction (Nouri, Nafchi, and Karim 2014).
In the flavor and fragrance industry, simultaneous distillation extraction (SDE) is usu-
ally considered superior to classical approaches such as solvent extraction or distillation.
The SDE technique was introduced and developed by Likens and Nickerson in 1964 for
volatile compounds in flavoring samples. This SDE technique has been successfully
applied for the extraction of aroma compounds, essential oils, and other volatile com-
pounds from various matrices (Chaintreau 2001; Khan et al. 2015; Khan et al. 2017).
As far as the authors know, SDE for the isolation of volatile compounds of betel
leaves has yet not been reported. Similarly, no report has been provided on the volatile
compound determination of betel leaves cultivated in Bangladesh. Therefore this study
was designed to characterize the volatile compounds present in five common varieties
of betel leaves from Bangladesh. The volatile compounds were isolated by the use of
SDE with 1:1 n-pentane:diethylether as the solvent and subsequent analysis by GC/MS.
Subsequently, principal component analysis (PCA) and hierarchical cluster analysis
(HCA) were used to classify the analyzed samples using the identified volatile com-
pounds as fingerprints. By comparing the composition of volatile compounds among
the Bangladeshi betel leaf varieties, attempts to characterize the medicinal properties of
each subject species are provided.

Materials and methods

Sample collection
Fresh betel leaves of five commonly consumed varieties of Bangladesh were collected in
triplicate. Out of these materials, 1 betel leaf sample BARI Paan 3 was collected from Spice
 ANALYTICAL LETTERS 2415

Table 1. Sample collection information on the five varieties of Piper betle L. from Bangladesh.
Symbol Scientific name Local name Origin Latitude and Longitude
A Piper betle L. var. bangla Bangla Paan Durgapur, Rajshahi, Bangladesh 24
290 270N and 88
420 190E
B Piper betle L. var. sanchi Sanchi Paan Durgapur, Rajshahi, Bangladesh 24
310 180N and 88
430 270E
C Piper betle L. var. misti Misti Paan Moheskhali, Chittagong, 21
310 100N and 91
570 520E
Bangladesh
D Piper betle L. var. khasia Khasia Paan Sirimongol, Sylhet, Bangladesh 24
090 220N and 91
440 370E
E Piper betle L. var. BARI Paan 3 BARI Paan 3 Spice research center, 24
580 420N and 89
200 150E
Sibgonj, Bogra, Bangladesh

Research Institute, Bogra, Bangladesh while other 4 common local varieties were collected
from specific shade garden with traditional farming system as shown in Table 1. For each
variety, fresh, healthy, green and mature betel leaves of different sizes were collected from
more than 200 plants in February 2019.
The samples were verified by a Bangladeshi Regional Agricultural Extension Field
Officer. After collection, the betel leaves were washed thoroughly in clean water, fol-
lowed by rinsing with distilled water. The clean leaves were subsequently dried at room
temperature for approximately 14 days. All dry leaves were ground into a powder using
a blender (MR 350CA, Braun, Spain) and stored at 4
C until analysis.

Chemicals and reagents

All chemicals and reagents were purchased from Fisher Scientific (Waltham,
Massachusetts, USA) and Sigma Company (St Louis, MO, USA).
Ultra-pure water was prepared with a purification system (Millepore Corporation,
Bedford, USA).
The n-pentane and diethyl ether organic solvents were redistilled using a wire spiral
packed double distillation apparatus (Normschliff Geratebau, Wertheim, Germany).
Anhydrous Na2SO4 was used for dehydration of organic solvents after heating over-
night at 650
C in a F 6000 furnace (Barnsted Thermolyne Company, IA, USA).

Extraction of volatile compounds

Each 10 g aliquot of powdered sample was mixed with 1 L of distilled water and the pH
was adjusted to 7.0 by adding dilute NaOH or HCl solution. Next, 10 mL of 110 ppm
n-butyl benzene in n-pentane was added as the internal standard for the quantitative
determination of the volatile compounds.
The volatile compounds were isolated from betel leaves for 3 hours of simultaneous
distillation extraction (SDE) (Likens and Nickerson) with 100 mL of redistilled 1:1 (v/v)
n-pentane:diethyl ether as described by Schultz et al. (1977). The procedure was contin-
ued under normal atmospheric pressure. The extract was dehydrated for 12 hours with
anhydrous Na2SO4 and concentrated to a final volume of approximately 1.5 mL using a
Vigreux column.
The samples were further concentrated to 0.5 mL by a stream of nitrogen. Lastly, the
concentrated extract was introduced into the GC/MS instrument for analysis (Khan
et al. 2017).
2416 M. A. ISLAM ET AL.

Determination of volatile compounds by GC/MS

An Agilent 7890 gas chromatograph (GC) coupled with an Agilent 7000 mass spectrom-
eter (MS) (Agilent Technologies, Santa Clara, CA, USA) in the electron impact (EI)
mode was used for the quantitative determination of the volatile compounds. The ion-
ization voltage, temperature of the injector and temperature of ion source were 70 eV,
250
C and 220
C, respectively. The mass spectra were scanned from 50 to 400 m/z.
A ZB-5MS capillary column (60 m  0.25 mm i.d., 0.25 mm film thickness,
Phenomenex, USA) was used for the separation of the volatiles. The oven temperature
was programed as follows: 40
C (isothermal for 3 min), increased to 220
C at 2
C
min1 and then to 300
C at 10
C min1 (isothermal for 10 min). Helium was used as
the carrier gas at a flow rate of 1.0 mL min1 with an injection volume of 1 mL using a
1:20 split ratio (Khan et al. 2017).

Identification and quantification of volatile compounds

The betel leaf volatile compounds were identified by our own mass spectral database
and the spectral libraries including FFNSC (2012), NIST 12 and WILEY 7 that were
provided with the GC/MS instrument and in mass spectral volumes (Davies 1990).
Additionally, comparisons of retention indices to reference data were considered
(Adams 2007). The linear retention index was used as a parameter by comparing the
retention times of solutes with those of standard n-alkanes (C8 through C22) as an
external reference (Jeon et al. 2017). The quantitative analysis of volatile compounds
was carried out by the use of the peak area percent of the internal standard employing
the following relationship:

C  1000
Volatile concentration mg=kg ¼ (1)
AB

where A is the peak area of each sample of the internal standard, B is the mass of the
sample (g), and C is the peak area of each component in the sample.

Statistical analysis
The volatile compounds were determined in triplicate. The results were evaluated using
Statistical Package for Social Sciences (SPSS), Software Version 20 (IBM, New York,
USA). The results were reported as mean ± standard deviation (mg/kg) on a dry
weight basis.
Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were
performed by the application of the PAST (version 3.25) software. The PCA and HCA
multivariate statistics were applied to better understand and differentiate the five vari-
eties of betel leaves analyzed by the characterization of the volatile compound
composition.
 ANALYTICAL LETTERS 2417

Figure 1. Comparison of GC-MS chromatograms of the volatile compounds in five varieties of Piper
betle L. from Bangladesh. Peak identification: I.S, internal standard; (1) 2-ethylfuran; (9) 1-hexanol; (14)
camphene; (26) eucalyptol; (32) linalool; (42) estragole; (45) chavicol; (52) eugenol; (58) b-caryophyl-
lene; (80) (E)-calamenene; (84) caryophyllene oxide; and (98) phytone.
Table 2. Volatile compounds identified in the five varieties of Piper betle L. from Bangladesh. The results are reported as the mean ± standard
2418

deviation (n ¼ 3).
BARI
Bangla Sanchi Misti Khasia Paan 3

Retention Retention Compound Molecular Molecular Content Content Content Content Content
Number time (min) index name formula weight Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg)
1 8.233 730 2-Ethylfuranf C6H8O 96 0.22 ± 0.00 9.41 ± 0.30 0.10 ± 0.00 7.17 ± 0.10 0.06 ± 0.00 7.76 ± 0.08 0.03 ± 0.00 2.79 ± 0.06 0.07 ± 0.00 8.50 ± 0.22
2 11.20 776 2-Penten-1-ola C5H10O 86 0.01 ± 0.00 0.27 ± 0.01 0.00 ± 0.00 0.23 ± 0.00 0.00 ± 0.00 0.44 ± 0.01 - - 0.00 ± 0.00 0.22 ± 0.00
3 12.73 799 3-Hexenalb C6H10O 98 0.05 ± 0.00 2.05 ± 0.05 0.01 ± 0.00 0.88 ± 0.08 0.02 ± 0.00 2.19 ± 0.08 0.01 ± 0.00 1.34 ± 0.25 0.02 ± 0.00 1.82 ± 0.12
4 12.83 800 Hexanalb C6H12O 100 0.12 ± 0.00 5.12 ± 0.06 0.08 ± 0.00 5.67 ± 0.51 0.03 ± 0.00 3.91 ± 0.05 0.03 ± 0.00 3.12 ± 0.08 0.03 ± 0.00 3.58 ± 0.11
M. A. ISLAM ET AL.

5 14.68 828 Furfuraldehydeb C5H4O2 96 0.01 ± 0.00 0.47 ± 0.01 0.01 ± 0.00 0.59 ± 0.01 0.01 ± 0.00 0.84 ± 0.02 0.00 ± 0.00 0.55 ± 0.01 - -
6 16.10 850 2-Hexenalb C6H10O 98 0.24 ± 0.00 10.29 ± 0.17 0.14 ± 0.00 0.00 ± 0.00 0.1 ± 0.00 14.03 ± 0.17 0.05 ± 0.00 6.06 ± 0.19 0.13 ± 0.00 15.34 ± 0.36
7 16.23 852 3-Hexen-1-ola C6H12O 100 0.15 ± 0.00 6.32 ± 0.10 0.06 ± 0.00 4.44 ± 0.07 0.03 ± 0.00 4.56 ± 0.08 0.01 ± 0.00 1.15 ± 0.04 0.04 ± 0.00 4.16 ± 0.20
8 16.93 863 2-Hexen-1-ola C6H12O 100 0.01 ± 0.00 0.44 ± 0.02 - - 0.00 ± 0.00 0.40 ± 0.01 - - - -
9 17.17 866 1-Hexanola C6H14O 102 0.04 ± 0.00 1.84 ± 0.03 0.02 ± 0.00 1.32 ± 0.02 0.01 ± 0.00 1.19 ± 0.02 0.01 ± 0.00 0.66 ± 0.42 0.01 ± 0.00 0.91 ± 0.05
10 20.15 910 (Z)22-Pentenyl acetatec C7H12O2 128 - - - - - - - - 0.02 ± 0.00 1.81 ± 0.06
11 20.91 920 Dimethylallyl acetatec C7H12O2 128 - - - - 0.00 ± 0.00 0.26 ± 0.01 - - - -
12 21.33 925 a-Thujened C10 H16 136 - - - - 0.01 ± 0.00 1.07 ± 0.01 - - - -
13 21.89 933 a-Pinened C10H16 136 0.09 ± 0.00 4.09 ± 0.05 0.05 ± 0.00 3.78 ± 0.05 0.15 ± 0.00 20.32 ± 0.15 0.16 ± 0.00 17.62 ± 0.66 0.04 ± 0.00 5.18 ± 0.10
14 23.16 950 Camphened C10H16 136 0.08 ± 0.00 3.56 ± 0.05 0.05 ± 0.00 3.45 ± 0.04 0.23 ± 0.00 32.68 ± 0.19 0.09 ± 0.00 10.37 ± 0.34 0.07 ± 0.00 8.64 ± 0.14
15 23.98 960 Benzaldehydeb C7H6O 106 0.01 ± 0.00 0.49 ± 0.01 0.01 ± 0.00 0.43 ± 0.00 0.01 ± 0.00 0.91 ± 0.03 0.00 ± 0.00 0.55 ± 0.01 0.01 ± 0.00 0.69 ± 0.03
16 25.28 977 b-Pinened C10H16 136 0.02 ± 0.00 0.79 ± 0.01 0.01 ± 0.00 0.48 ± 0.01 0.02 ± 0.00 2.96 ± 0.07 0.03 ± 0.00 3.33 ± 0.29 - -
17 25.73 983 Methyl heptenone C8H14O 126 0.01 ± 0.00 0.61 ± 0.01 0.01 ± 0.00 0.69 ± 0.01 0.00 ± 0.00 0.43 ± 0.01 0.01 ± 0.00 1.19 ± 0.04 0.00 ± 0.00 0.49 ± 0.03
18 26.19 989 2-pentylfuranf C9H14O 138 0.04 ± 0.00 1.79 ± 0.02 0.02 ± 0.00 1.65 ± 0.02 - - - - - -
19 26.21 990 Dehydrocineolef C10H16O 152 - - - - - - 0.04 ± 0.00 4.88 ± 0.20 - -
20 27.30 1004 (Z)3-Hexenyl-1-acetatec C8H14O2 142 0.22 ± 0.00 9.50 ± 0.15 0.13 ± 0.00 9.25 ± 0.14 0.05 ± 0.00 6.84 ± 0.11 0.06 ± 0.00 6.75 ± 0.27 0.10 ± 0.00 11.92 ± 0.24
21 27.86 1011 n-Hexyl acetatec C6H16O2 100 0.09 ± 0.00 3.88 ± 0.07 0.05 ± 0.00 3.42 ± 0.04 0.02 ± 0.00 2.18 ± 0.03 0.04 ± 0.00 3.92 ± 0.16 0.02 ± 0.00 2.54 ± 0.08
22 28.05 1014 (E)2-Hexenyl acetatec C8H14O2 142 0.01 ± 0.00 0.38 ± 0.01 0.01 ± 0.00 0.38 ± 0.01 0.00 ± 0.00 0.33 ± 0.00 0.00 ± 0.00 0.42 ± 0.02 0.00 ± 0.00 0.46 ± 0.02
23 28.30 1017 4-d-carene C10H16 136 - - - - - - 0.00 ± 0.00 0.49 ± 0.01 - -
24 28.89 1025 P-cymened C10H14 134 0.01 ± 0.00 0.26 ± 0.01 - - 0.00 ± 0.00 0.68 ± 0.01 0.01 ± 0.00 1.01 ± 0.02 0.00 ± 0.00 0.45 ± 0.01
25 29.28 1029 Limonened C10H16 136 0.31 ± 0.00 13.27 ± 0.16 0.18 ± 0.00 12.58 ± 0.15 0.24 ± 0.00 33.56 ± 0.19 0.07 ± 0.00 7.39 ± 0.23 0.18 ± 0.00 21.58 ± 0.39
26 29.52 1033 Eucalyptolf C10H18O 154 0.18 ± 0.00 7.91 ± 0.10 0.11 ± 0.00 8.00 ± 0.11 0.73 ± 0.00 102.4 ± 0.7 1.32 ± 0.01 146.6 ± 3.9 0.15 ± 0.00 17.55 ± 0.29
27 30.32 1043 Phenyl acetaldehydeb C8H8O 120 0.05 ± 0.00 2.39 ± 0.02 0.03 ± 0.00 1.82 ± 0.06 0.05 ± 0.00 6.64 ± 0.19 0.03 ± 0.00 2.84 ± 0.09 0.03 ± 0.00 2.93 ± 0.11
28 30.56 1046 (E)-Ocimeneg C10H16 136 - - - - - - - - 0.00 ± 0.00 0.29 ± 0.01
29 31.03 1052 2,3-Butanediyl diacetatec C8H14O4 174 - - - - - - 0.07 ± 0.00 7.43 ± 0.29 0.01 ± 0.00 1.70 ± 0.05
I.S. 31.35 1056 n-Butyl benzene C9H14 134 - - - - - - - - - -
(Internal standard)
30 31.52 1058 c-Terpinened C10H16 136 - - - - - - 0.01 ± 0.00 0.61 ± 0.03 - -
31 32.45 1071 (Z)-Sabinene hydratea C10H18O 154 - - - - 0.00 ± 0.00 0.49 ± 0.01 0.01 ± 0.00 0.89 ± 0.04 - -
32 34.68 1099 Linaloola C10H18O 154 0.66 ± 0.01 28.53 ± 0.65 0.31 ± 0.00 21.9 ± 0.40 0.25 ± 0.00 34.76 ± 0.42 1.12 ± 0.02 124.0 ± 4.4 - -
33 35.04 1104 Nonanalb C9H18O 142 0.03 ± 0.00 1.40 ± 0.03 0.03 ± 0.00 1.86 ± 0.04 0.01 ± 0.00 1.17 ± 0.03 0.01 ± 0.00 0.86 ± 0.01 0.01 ± 0.00 0.95 ± 0.03
34 35.56 1111 Phenylethyl alcohola C8H10O 122 0.01 ± 0.00 0.38 ± 0.02 0.00 ± 0.00 0.24 ± 0.02 0.00 ± 0.00 0.37 ± 0.03 0.00 ± 0.00 0.17 ± 0.01 - -
35 38.04 1144 Oxophoronee C9H12O2 152 0.01 ± 0.00 0.26 ± 0.01 - - - - - - - -
36 38.21 1146 (E)-Verbenola C10H16O 152 - - - - - - 0.01 ± 0.00 0.62 ± 0.05 - -
37 39.35 1162 Benzyl acetatec C9H10O2 150 0.01 ± 0.00 0.39 ± 0.01 0.02 ± 0.00 1.05 ± 0.01 0.00 ± 0.00 0.57 ± 0.12 0.04 ± 0.00 4.81 ± 0.24 0.01 ± 0.00 1.02 ± 0.04
38 40.86 1182 Terpinen-4-ola C10H18O 154 - - - - 0.01 ± 0.00 0.79 ± 0.00 - - - -
39 41.25 1187 p-Cymen-8-ola C10H14O 150 - - - - - - 0.01 ± 0.00 0.81 ± 0.04 - -
40 41.63 1192 Methyl salicylatec C8H8O3 152 0.03 ± 0.00 1.17 ± 0.02 0.04 ± 0.00 2.66 ± 0.01 0.01 ± 0.00 1.27 ± 0.01 0.06 ± 0.00 6.63 ± 0.31 0.02 ± 0.00 2.08 ± 0.13
41 41.92 1196 a-Terpineola C10H18O 154 - - - - 0.00 ± 0.00 0.14 ± 0.00 0.04 ± 0.00 4.62 ± 0.16 - -
42 42.08 1198 Estragolef C10H12O 148 0.08 ± 0.00 3.34 ± 0.07 0.06 ± 0.00 4.08 ± 0.08 0.75 ± 0.01 104.39 ± 1.62 0.13 ± 0.00 14.26 ± 0.78 0.08 ± 0.00 9.82 ± 0.18
43 42.62 1206 Decanalb C10H20O 156 0.04 ± 0.00 1.88 ± 0.06 0.04 ± 0.00 2.70 ± 0.06 0.02 ± 0.00 2.45 ± 0.03 0.01 ± 0.00 1.60 ± 0.09 0.04 ± 0.00 4.15 ± 0.12
44 43.65 1220 b-Cyclocitralb C10H16O 152 0.01 ± 0.00 0.51 ± 0.01 0.01 ± 0.00 0.65 ± 0.01 - - 0.01 ± 0.00 0.57 ± 0.03 0.01 ± 0.00 0.73 ± 0.00
45 45.76 1250 Chavicola C9H10O 134 0.26 ± 0.00 11.39 ± 0.23 0.61 ± 0.01 42.18 ± 0.79 11.95 ± 0.08 1667 ± 16 6.08 ± 0.08 675.1 ± 18.7 3.97 ± 0.03 463.5 ± 6.5
46 46.05 1254 2-Phenylethyl acetatec C10H12O2 164 0.02 ± 0.00 0.97 ± 0.02 0.02 ± 0.00 1.39 ± 0.03 0.01 ± 0.00 1.71 ± 0.03 0.02 ± 0.00 2.76 ± 0.11 0.01 ± 0.00 1.18 ± 0.06
47 47.93 1280 Linalool oxide acetatec C12H20O3 212 0.05 ± 0.00 2.00 ± 0.05 0.01 ± 0.00 0.57 ± 0.02 0.02 ± 0.00 2.41 ± 0.03 - - - -
48 48.70 1291 1-H-Indold C8H7N 117 - - 0.03 ± 0.00 2.16 ± 0.06 - - - - - -
49 48.78 1292 Undecan-2-onee C11H22O 170 - - - - - - - - 0.02 ± 0.00 2.54 ± 0.04
50 49.96 1309 4-vinyl Guaiacola C9H10O2 150 0.01 ± 0.00 0.53 ± 0.01 - - 0.04 ± 0.03 6.18 ± 1.14 - - - -
51 52.12 1341 4-Allylphenyl acetatec C11H12O2 176 - - - - - - 0.01 ± 0.00 1.41 ± 0.05 - -
52 54.16 1372 Eugenola C10H12O2 164 78.52 ± 0.27 3413 ± 1 85.18 ± 0.15 5927 ± 17 72.3 ± 0.22 10092 ± 0.2 67.73 ± 0.63 7524 ± 42 81.84 ± 0.12 9563 ± 159
53 55.68 1394 b-Elemened C15H24 204 0.6 ± 0.01 26.24 ± 0.56 0.27 ± 0.00 18.59 ± 0.43 0.05 ± 0.00 7.46 ± 0.03 0.33 ± 0.00 36.21 ± 0.75 0.09 ± 0.00 10.13 ± 0.17
54 56.08 1400 Methyleugenold C11H14O2 178 0.81 ± 0.01 35.18 ± 0.69 0.64 ± 0.01 44.58 ± 0.99 0.96 ± 0.00 133.93 ± 0.84 0.52 ± 0.02 57.67 ± 3.08 0.43 ± 0.01 50.33 ± 0.82
55 56.76 1411 n-Decyl acetatec C12H24O2 200 - - - - - - - - 0.03 ± 0.00 3.95 ± 0.05
56 56.73 1410 n-Dodecanalb C12H24O 184 0.19 ± 0.00 8.32 ± 0.20 0.18 ± 0.00 12.39 ± 0.23 - - 0.07 ± 0.00 7.33 ± 0.46 0.09 ± 0.00 10.56 ± 0.23
57 57.11 1416 (Z)-a- Bergamotened C15H24 204 0.17 ± 0.00 7.26 ± 0.18 0.12 ± 0.00 8.00 ± 0.11 0.02 ± 0.00 2.66 ± 0.06 0.49 ± 0.01 54.48 ± 2.60 0.1 ± 0.00 11.14 ± 0.04
58 57.70 1426 b-Caryophyllened C15 H24 204 2.64 ± 0.04 114.7 ± 2.2 1.74 ± 0.01 121.4 ± 1.4 4.24 ± 0.06 592.4 ± 10.5 4.93 ± 0.03 547.8 ± 11.2 4.69 ± 0.04 547.4 ± 3.0
59 58.23 1434 c-Elemened C15 H24 204 - - - - - - 0.01 ± 0.00 1.28 ± 0.36 - -
60 58.53 1439 a-Guaiened C15H24 204 0.01 ± 0.00 0.56 ± 0.01 0.01 ± 0.00 0.57 ± 0.00 0.02 ± 0.00 3.42 ± 0.03 0.57 ± 0.01 63.77 ± 2.67 0.02 ± 0.00 1.77 ± 0.01
61 58.85 1444 Aromadendrened C15H24 204 - - 0.01 ± 0.00 0.57 ± 0.01 0.01 ± 0.00 1.50 ± 0.02 0.03 ± 0.00 2.97 ± 0.05 0.03 ± 0.00 3.43 ± 0.05
62 58.83 1445 9-epi-b-Caryophyllened C15H24 204 0.01 ± 0.00 0.51 ± 0.04 - - - - - - - -
63 59.18 1449 Isogermacrene Dd C15H24 204 0.18 ± 0.00 7.94 ± 0.18 0.12 ± 0.00 8.33 ± 0.18 0.05 ± 0.00 6.85 ± 0.08 0.11 ± 0.00 12.22 ± 0.64 0.07 ± 0.00 7.87 ± 0.09
64 59.54 1455 Sesquisabinened C15H24 204 - - - - - - 0.13 ± 0.00 14.86 ± 0.70 0.06 ± 0.00 7.25 ± 0.12
65 59.93 1461 a-Humulened C15H24 204 1.35 ± 0.02 58.77 ± 1.25 0.87 ± 0.00 60.57 ± 0.55 0.95 ± 0.00 132.63 ± 1.13 - - 1.03 ± 0.00 120.0 ± 1.9
66 60.05 1463 (Z)-a-Bisabilene C15H24 204 - - 0.05 ± 0.00 3.76 ± 0.09 - - 0.21 ± 0.00 23.13 ± 1.21 - -
67 60.86 1475 Cadina-1(6),4-diened C15H24 204 0.02 ± 0.00 0.69 ± 0.03 0.02 ± 0.00 1.62 ± 0.11 0.05 ± 0.00 6.46 ± 0.08 - - 0.06 ± 0.00 7.06 ± 0.14
68 61.14 1480 c-Muurolened C15H24 204 6.45 ± 0.10 280.4 ± 5.4 4.49 ± 0.04 312.5 ± 4.4 0.03 ± 0.00 3.50 ± 0.13 1.74 ± 0.06 193.2 ± 9.4 0.07 ± 0.00 8.09 ± 0.14
69 61.21 1481 Valencened C15H24 204 - - 0.22 ± 0.00 15.46 ± 0.23 3.02 ± 0.04 421.66 ± 7.33 6.18 ± 0.01 686.7 ± 8.43 3.11 ± 0.02 362.85 ± 4.72
70 61.32 1483 a-Curcumened C15H22 202 0.31 ± 0.00 13.31 ± 0.36 0.05 ± 0.00 3.65 ± 0.06 - - 0.68 ± 0.00 75.59 ± 2.00 0.16 ± 0.00 18.56 ± 0.26
71 61.58 1487 b-(Z)-Bergamotened C15H24 204 0.09 ± 0.00 3.90 ± 0.10 0.02 ± 0.00 1.08 ± 0.03 - - 0.23 ± 0.00 25.61 ± 1.03 - -
72 61.89 1492 Eremophilened C15H24 204 0.02 ± 0.00 0.97 ± 0.04 – – 0.01 ± 0.00 1.55 ± 0.02 0.06 ± 0.00 6.61 ± 0.96 - -
73 62.02 1494 b-Selinened C15H24 204 0.07 ± 0.00 2.86 ± 0.03 0.04 ± 0.00 2.94 ± 0.06 0.05 ± 0.00 7.64 ± 0.14 0.48 ± 0.04 53.3 ± 5.60 0.12 ± 0.00 14.11 ± 0.20
74 62.19 1496 c-Amorphened C15H24 204 0.05 ± 0.00 1.98 ± 0.06 0.04 ± 0.00 2.91 ± 0.03 0.03 ± 0.00 4.34 ± 0.01 0.21 ± 0.01 22.94 ± 1.98 0.09 ± 0.00 10.25 ± 0.25
75 62.37 1499 Cubebola C15H26O 222 0.42 ± 0.01 18.44 ± 0.61 0.18 ± 0.00 12.87 ± 0.35 0.24 ± 0.07 33.48 ± 10.9 - - - -
76 62.48 1501 a-Muurolened C15H24 204 0.36 ± 0.01 15.83 ± 0.53 0.27 ± 0.00 18.66 ± 0.55 - - - - - -
77 62.51 1501 a-Amorphened C15H24 204 - - - - 0.13 ± 0.00 18.55 ± 0.78 - - 0.45 ± 0.00 52.51 ± 0.72
78 63.52 1518 Eugenyl acetatec C12H14O3 206 1.50 ± 0.00 65.38 ± 0.60 0.68 ± 0.01 47.29 ± 1.10 0.92 ± 0.00 127.82 ± 0.19 - - 0.19 ± 0.00 22.31 ± 0.23
79 63.78 1523 d-Cadinened C15H24 204 0.35 ± 0.00 15.24 ± 0.37 0.36 ± 0.00 24.87 ± 0.62 0.2 ± 0.00 27.65 ± 1.19 1.75 ± 0.07 194.4 ± 10.9 - -
80 63.91 1525 (E)-Calamenened C15H22 202 0.24 ± 0.00 10.42 ± 0.06 0.26 ± 0.00 17.77 ± 0.40 0.14 ± 0.00 19.00 ± 0.02 0.66 ± 0.01 73.64 ± 2.39 0.68 ± 0.00 79.27 ± 1.45
ANALYTICAL LETTERS

81 64.58 1536 (E)-Cadina-1,4-diened C15H24 204 0.03 ± 0.00 1.50 ± 0.03 0.04 ± 0.00 2.49 ± 0.05 0.02 ± 0.00 2.88 ± 0.02 0.12 ± 0.00 13.30 ± 0.69 0.05 ± 0.00 5.84 ± 0.10
82 66.14 1562 a-Nerolidola C15H26O 222 - - - - 0.03 ± 0.00 4.86 ± 0.07 0.14 ± 0.03 16.06 ± 4.40 0.02 ± 0.00 2.82 ± 0.03
83 66.18 1563 1-nor-Bourbonanonee C14H22O 206 0.09 ± 0.00 4.05 ± 0.09 0.03 ± 0.00 2.08 ± 0.07 - - - - - -
(continued)
2419
Table 2. Continued.
2420

BARI
Bangla Sanchi Misti Khasia Paan 3

Retention Retention Compound Molecular Molecular Content Content Content Content Content
Number time (min) index name formula weight Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg) Area (%) (mg/kg)
84 67.69 1588 Caryophyllene oxidef C15H24O 220 1.34 ± 0.01 58.26 ± 0.97 0.80 ± 0.23 55.96 ± 16.50 1.17 ± 0.01 163.0 ± 2.9 1.03 ± 0.04 114.3 ± 6.4 0.71 ± 0.00 82.8 ± 0.81
85 68.26 1597 Salvial-4(14)-en-1-onee C15H24O 220 0.03 ± 0.00 1.27 ± 0.06 0.01 ± 0.01 0.97 ± 0.77 - - 0.03 ± 0.00 2.84 ± 0.29 0.01 ± 0.00 0.98 ± 0.02
86 69.16 1613 Tetradecanalb C14H28O 212 0.11 ± 0.00 4.93 ± 0.08 0.11 ± 0.00 7.56 ± 0.36 - - - - 0.10 ± 0.00 12.1 ± 0.21
87 69.29 1615 Humulene epoxide IIf C15H24O 220 0.39 ± 0.00 17.12 ± 0.35 0.29 ± 0.00 20.01 ± 0.32 0.18 ± 0.00 24.63 ± 0.52 0.28 ± 0.01 30.94 ± 1.57 0.03 ± 0.00 4.06 ± 0.05
88 70.25 1632 Epicubenola C15H26O 222 - - - - 0.09 ± 0.00 12.92 ± 0.21 - - 0.13 ± 0.00 15.43 ± 0.06
M. A. ISLAM ET AL.

89 70.56 1637 b-Spathulenola C15H24O 220 - - - - - - 0.15 ± 0.00 16.49 ± 1.16 - -

90 71.28 1650 a-Muurolola C15H26O 222 0.02 ± 0.00 0.69 ± 0.04 0.11 ± 0.00 7.63 ± 0.07 0.05 ± 0.00 7.08 ± 0.06 0.36 ± 0.03 39.8 ± 3.99 0.11 ± 0.00 12.64 ± 0.11
91 71.80 1659 Cadin-4-en-10-ola C15H26O 222 0.12 ± 0.00 5.25 ± 0.09 0.19 ± 0.00 13.23 ± 0.13 - - 0.34 ± 0.01 38.01 ± 2.40 0.12 ± 0.00 14.20 ± 0.17
92 71.96 1662 Pogostola C15H26O 222 - - - - 0.07 ± 0.00 10.11 ± 0.10 - - - -
93 72.52 1672 b-Bisabolola C15H26O 222 0.09 ± 0.00 4.07 ± 0.07 0.07 ± 0.00 5.17 ± 0.09 - - 0.37 ± 0.03 40.86 ± 4.53 0.14 ± 0.00 16.29 ± 0.21
94 72.76 1676 Cadalened C15H18 198 0.01 ± 0.00 0.44 ± 0.03 0.06 ± 0.00 3.84 ± 0.04 0.05 ± 0.00 6.89 ± 0.18 0.21 ± 0.00 23.12 ± 1.05 - -
95 76.73 1744 Guaiac acetatec C17H28O2 264 - - - - - - 0.14 ± 0.00 15.99 ± 1.23 - -
96 76.72 1748 (E)-Cadinola C15H26O 222 0.11 ± 0.01 4.75 ± 0.65 0.12 ± 0.00 8.17 ± 0.12 0.04 ± 0.00 5.26 ± 0.21 0.03 ± 0.00 2.82 ± 0.16 0.03 ± 0.00 3.47 ± 0.02
97 79.84 1806 Farnesyl acetatec C17H28O2 264 0.01 ± 0.00 0.37 ± 0.01 0.01 ± 0.00 0.62 ± 0.01 0.01 ± 0.00 1.42 ± 0.03 0.04 ± 0.00 4.87 ± 0.32 0.01 ± 0.00 1.14 ± 0.03
98 81.59 1840 Phytonee C18H36O 268 0.07 ± 0.00 3.07 ± 0.06 0.04 ± 0.00 2.65 ± 0.06 0.01 ± 0.00 1.44 ± 0.05 0.02 ± 0.00 1.70 ± 0.11 0.01 ± 0.00 0.95 ± 0.02
99 85.08 1909 Farnesyl acetonee C18H30O 262 0.02 ± 0.00 0.75 ± 0.03 0.03 ± 0.00 2.09 ± 0.01 - - - - - -
100 86.86 1945 Isophytola C20H40O 296 0.02 ± 0.00 0.69 ± 0.01 0.01 ± 0.00 0.93 ± 0.02 0.01 ± 0.00 0.81 ± 0.03 0.01 ± 0.00 0.96 ± 0.10 0.01 ± 0.00 0.65 ± 0.02
101 99.66 2205 Octadecanol C20H40O2 312 - - - - 0.01 ± 0.00 2.04 ± 0.04 - - 0.02 ± 0.00 2.55 ± 0.06
acetatec
Total 100.00 4347 100.00 6959 100.00 13959 100.00 11091 100.00 11487
0.00 designates a value less than 0.01 and - represents not detected.
a-k
The superscripts represent the functional group of compound where a represents alcohols; b represents aldehydes; c represents esters; d represents hydrocarbons; e represents
ketones; f represents ethers; and g represents miscellaneous.
Compound names in bold represent the first reported of volatile compounds in betel leaves.
 ANALYTICAL LETTERS 2421

Results
The GC/MS chromatograms of the five betel leaf varieties are shown in Figure 1. The con-
centrations of the identified volatile compounds details are provided in Table 2. In general,
the essential oil from Piper betle L. leaf was shown to contain a variety of volatile com-
pounds having a wide range of bioactivities. In the literature, it has been reported that
betel leaves contain between 40 and 50 volatile compounds (Basak and Guha 2015).
In the present study, 67 to 74 volatile compounds were identified in the five varieties of
betel leaf. This number is the highest compared to the previous reported studies (Alighiri
et al. 2018; Karak et al. 2018). A total of 101 volatile compounds were identified in the ana-
lyzed betel leaves. To the best of our knowledge, 50 new betel leaf volatile compounds
were reported for the first time in this study (Table 2). The total volatile compound con-
centrations on a dry weight basis varied from 4347 mg/kg to 13959 mg/kg (Table 2).

Volatile compounds in Piper betle L. var. Bangla

As shown in Table 3, this study confirms that the total concentration of volatile com-
pounds was 4347 mg/kg in Bangla. 72 compounds from different chemical functional
groups were identified, including 24 hydrocarbons (13.47%, 585.4 mg/kg), 15 alcohols
(80.44%, 3497 mg/kg), 11 aldehydes (0.87%, 437.8 mg/kg), 9 esters (1.93%, 84.02 mg/kg),
7 ethers (3.06%, 133.0 mg/kg) and 6 ketones (0.23%, 10.00 mg/kg). The terpene group
was the dominant chemical class with the highest proportion (95.52%, 4152 mg/kg), fol-
lowed by the oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes,
monoterpene hydrocarbons and oxygenated diterpenes at (79.51%, 3456 mg/kg),
(12.96%, 563.5 mg/kg), (2.53%, 109.8 mg/kg), (0.51%, 21.96 mg/kg) and (0.02%, 0.69 mg/
kg), respectively (Table 4). The major volatile compounds were eugenol (78.52%,
3413 mg/kg) followed by c-muurolene (6.45%, 280.4 mg/kg), b-caryophyllene (2.64%,
114.7 mg/kg), eugenol acetate (1.50%, 65.38 mg/kg), a-hemulene (1.35%, 58.77 mg/kg)
and caryophyllene oxide (1.34%, 58.26 mg/kg).

Two compounds, oxophorone and 9-epi-b-caryophyllene were found only in the

Bangla betel leaves which differentiate this plant from the others (Table 2).

Volatile compounds in Piper betle L. var. Sanchi

In Table 3, the total concentration of volatile compounds in the Sanchi betel leaves was
shown to be 6959 mg/kg. These were a total of 70 volatile compounds identified, includ-
ing 24 hydrocarbons (9.34%, 649.9 mg/kg), 13 alcohols (86.88%, 6046 mg/kg), 11 alde-
hydes (0.64%, 44.34 mg/kg), 9 esters (0.96%, 66.63 mg/kg), 7 ethers (2.03%, 141.45 mg/
kg), 5 ketones (0.12%, 8.47 mg/kg) and one N-containing compound (0.03%, 2.16 mg/
kg). As shown in Table 4, terpenes (96.47%, 6740.80 mg/kg) were the most abundant
group followed by oxygenated monoterpenes (85.74%, 5966 mg/kg), sesquiterpene
hydrocarbons (9.05%, 629.6 mg/kg), oxygenated sesquiterpenes (1.78%, 124.0 mg/kg),
monoterpene hydrocarbons (0.29%, 20.29 mg/kg) and oxygenated diterpenes (0.01%,
0.93 mg/kg). The primary volatile compounds were eugenol (85.18%, 5927.30 mg/kg) fol-
lowed by c-muurolene (4.49%, 312.5 mg/kg) and b-caryophyllene (1.74%, 121.4 mg/kg).
 2422
M. A. ISLAM ET AL.

Table 3. Relative content of functional group detected in five varieties of Piper betle L. from Bangladesh.
Bangla Sanchi Misti Khasia BARI Paan-3
Functional Number of Area Content Number of Area Content Number of Area Content Number of Area Content Number of Area Content
Number group Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg)
1 Alcohol 15 80.44 3497 13 86.88 6046 19 85.13 11884 17 76.39 8487 12 86.41 10097
2 Aldehyde 11 0.87 37.82 11 0.64 44.34 8 0.23 32.14 10 0.22 24.82 10 0.45 52.85
3 Ester 9 1.93 84.02 9 0.96 66.63 11 1.05 146.8 10 0.49 54.99 12 0.45 52.66
4 Hydrocarbon 24 13.47 585.4 24 9.34 649.9 24 9.73 1358 27 19.49 2166 22 11.16 1304
5 Ketone 6 0.23 10.00 5 0.12 8.47 2 0.01 1.86 3 0.05 5.72 4 0.04 4.96
6 Ether 7 3.06 133.0 7 2.03 141.4 6 3.84 536.0 7 3.34 371.4 7 1.48 173.1
7 Miscellaneous 0 0 0 1 0.03 2.16 0 0 0 0 0 0 0 0 0
Total 72 100.00 4347 70 100.00 6959 70 100.00 13959 74 100.00 11110 67 100.00 11684
 ANALYTICAL LETTERS 2423

This variety was demonstrated to contain 1-H-indole as a unique compound that

may considered to be a marker for its identification (Table 2).

Volatile compounds in Piper betle L. var. Misti

The total concentration of volatile compounds in Misti betel leaf was 13959 mg/kg.
There were a total of 70 identified volatile compounds (Table 3), belonging to chemical
classes that included hydrocarbons (9.73%, 1359 mg/kg), alcohols (85.13%, 11884 mg/
kg), esters (1.05%, 146.8 mg/kg), aldehydes (0.23%, 32.14 mg/kg), ethers (3.84%,
536.0 mg/kg) and ketones (0.01%, 1.86 mg/kg). The terpenes (85.69%, 11962 mg/kg)
were the primary class of volatile compounds in Misti betel leaves. Oxygenated mono-
terpenes (74.07%, 10340 mg/kg), sesquiterpene hydrocarbons (9.08%, 1267 mg/kg), oxy-
genated sesquiterpenes (1.87%, 261.3 mg/kg), monoterpene hydrocarbons (0.65%,
91.27 mg/kg) and oxygenated diterpene (0.02%, 2.85 mg/kg) were also quantified (Table
4). The major volatile compounds were eugenol (72.30%, 10092 mg/kg), followed by
chavicol (11.95%, 1667 mg/kg), b-caryophyllene (4.24%, 592.4 mg/kg), valencene (3.02%,
421.7 mg/kg), and caryophyllene oxide (1.17%, 163.0 mg/kg).
Misti betel leaves were reported to have four unique volatile compounds that
included pogostol, a-thujene, terpene-4-ol and dimethylallyl acetate compared to the
other varieties (Table 2).

Volatile compounds in Piper betle L. var. Khasia

As mentioned in Table 3, a total of 74 volatile compounds were identified in the Khasia
betel leaves at a concentration equal to 11110 mg/kg. These were 27 hydrocarbons
(19.49%, 2166 mg/kg), 17 alcohols (76.39%, 8487 mg/kg), 10 aldehydes (0.22%, 24.82 mg/
kg), 10 esters (0.49%, 54.99 mg/kg), 7 ethers (3.34%, 371.44 mg/kg) and 3 ketones (0.05%,
5.72 mg/kg). Terpene compounds were the most abundant (92.66%, 10294 mg/kg) in kha-
sia betel leaves, followed by oxygenated monoterpenes (70.44%, 7825 mg/kg), sesquiter-
pene hydrocarbons (19.13%, 2125 mg/kg), oxygenated sesquiterpenes (2.72%, 302.1 mg/
kg), monoterpene hydrocarbons (0.37%, 40.82 mg/kg) and oxygenated diterpenes (0.01%,
0.96 mg/kg) (Table 4). The volatile compounds at the highest concentrations were eugenol
(67.73%, 7524 mg/kg), followed by valencene (6.18%, 686.7 mg/kg), chavivol (6.08%,
675.1 mg/kg), b-caryophyllene (4.93%, 547.8 mg/kg), d-cadenene (1.75%, 194.4 mg/kg),
c-muurolene (1.74%, 193.2 mg/kg), eucalyptol (1.32%, 146.6 mg/kg), linalool (1.12%,
124.0 mg/kg) and caryophyllene oxide (1.03%, 114.3 mg/kg).
The Khasia variety was demonstrated to contain the largest number of unique com-
pounds including dehydrocineole, 4-d-carene, c-terpinene, (E)-verbenol, p-cymen-8-ol, 4-
allylphenyl acetate and b-sapthulenol compared to the other betel leaf varieties (Table 2).

Volatile compounds in Piper betle L. var. BARI Paan 3

The analyses demonstrated that the yield of volatile compounds in BARI Paan 3 was
11684 mg/kg on a dry weight basis. There were a total of 67 compounds belonging to
various chemical classes including hydrocarbons (11.16%, 1304 mg/kg), alcohols
 2424
M. A. ISLAM ET AL.

Table 4. Relative content of terpene group detected in five varieties of Piper betle L. from Bangladesh.
Bangla Sanchi Misti Khasia BARI Paan-3
Number of Area Content Number of Area Content Number of Area Content Number of Area Content Number of Area Content
Number Class of terpenes Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg) Compounds (%) (mg/kg)
1 Monoterpene hydrocarbons 5 0.51 21.96 4 0.29 20.29 6 0.65 91.27 7 0.37 40.82 5 0.31 36.15
2 Oxygenated monoterpenes 7 79.51 3456 7 85.74 5966 9 74.07 10340 12 70.44 7825 6 82.13 9596
3 Sesquiterpene hydrocarbons 19 12.96 563.5 20 9.05 630 18 9.08 1267 20 19.13 2125 17 10.85 1268
4 Oxygenated sesquiterpenes 8 2.53 109.8 8 1.78 124.0 8 1.87 261.3 9 2.72 302.1 9 1.31 152.7
5 Oxygenated diterpenes 1 0.02 0.69 1 0.01 0.93 2 0.02 2.85 1 0.01 0.96 2 0.03 3.20
Total 40 95.52 4152 40 96.87 6741 43 85.69 11962 49 92.66 10294 39 94.62 11056
 ANALYTICAL LETTERS 2425

(86.41%, 10097 mg/kg), esters (0.45%, 52.66 mg/kg), aldehydes (0.45%, 52.85 mg/kg),
ethers (1.48%, 173.1 mg/kg) and ketones (0.04%, 4.96 mg/kg) (Table 3). The terpene
group was the dominant chemical class with largest portion of compounds (94.62%,
11056 mg/kg), followed by oxygenated monoterpenes (82.13%, 9596 mg/kg), sesquiter-
pene hydrocarbons (10.85%, 1268 mg/kg), oxygenated sesquiterpenes (1.31%, 152.7 mg/
kg), monoterpene hydrocarbons (0.31%, 36.15 mg/kg) and oxygenated diterpenes
(0.03%, 3.20 mg/kg) (Table 4). The primary volatile compounds were eugenol (81.84%,
9563 mg/kg), followed by b-caryophyllene (4.69%, 547.4 mg/kg), chavicol (3.97%,
463.5 mg/kg), valencene (3.11%, 362.8 mg/kg) and a-hemulene (1.03%, 120.1 mg/kg).
BARI Paan 3 was shown to contain four unique compounds including (Z)2-pentenyl
acetate, (E)-ocimene, n-decyl acetate and undecane-2-one that were not present in the
other varieties (Table 2).

Principal component analysis (PCA) and hierarchical cluster analysis (HCA)

To determine the similarity and to classify the betel leaf varieties based on the identified
volatile compounds, principal component analysis (PCA) and hierarchical cluster ana-
lysis (HCA) were applied. PCA was used to decrease the dimension of the data variance,
which was calculated based on the correlation of the contents of volatile compounds. In
the principal component analysis, there were two eigenvalues higher than 1, which
meant that there were two principal components for data analysis following the reduc-
tion of the dimensions.
Two principal components, i.e., PC1 (96.9%) and PC2 (2.2%) were used in order to
simplify the statistical analysis and to obtain a planer score plot of the principal compo-
nents. According to Figure 2, these components together explain 99.1% of the data vari-
ance although some information about the volatile compounds has been lost during the
statistical re-modeling. Of the volatile compounds, eugenol and chavicol had the highest
correlation values based on loadings for PC1 equal to 0.974 and 0.191, respectively. On
the other hand, chavicol and valencene had the maximum correlation values based on
the loading of PC2 at values of 0.947 and 0.139, individually.
The analyzed betel leaves formed three clusters in the score plot of principal compo-
nent analysis shown in Figure 2. Misti and BARI Paan 3 formed cluster I, Bangla pro-
duced cluster II, and Sanchi and Khasia formed cluster III.
Hierarchical cluster analysis was also applied based on the concentrations of the 101
identified volatile compounds. Ward’s method was used between group linkage and the
similarity between clusters was reported as the proximate analysis.
The five varieties of betel leaf also formed three clusters in the dendrogram (Figure 3).
Misti and BARI Paan 3 betel leaves formed cluster I, Bangla betel leaf provided cluster II,
and Sanchi and Khasia betel leaf varieties formed cluster III.

Discussion
Simultaneous distillation extraction (SDE) is widely known to be one of the prominent
methods for isolating volatile compounds from plant samples (Jeon et al. 2017; Majcher
and Jele
n 2009; Khan et al. 2015). In this work, SDE was successfully applied to isolate
2426 M. A. ISLAM ET AL.

Figure 2. Score plot of two principal components analysis in five varieties of Piper betle L.
from Bangladesh.

Figure 3. Dendogram for the volatile compounds in five varieties of Piper betle L. from Bangladesh.

the volatile compounds in betel leaves that were subsequently analyzed by GC/MS.
Differences in the composition of volatile compounds was observed among the betel
leaf varieties.
Tables 2 and 3 show that the alcohols were the most commonly found functional
group of volatile compounds identified in the studied betel leaf varieties, along with the
phenolic compounds. Additionally, terpene compounds were the dominant chemical
compounds in all five varieties of betel leaves, for which the concentrations of oxygen-
ated monoterpene compounds were the highest, as shown in Table 4.
Bajpai et al. (2010) reported that the primary groups of volatile compounds in betel
leaves are terpenes and phenols, and thus the current research findings correspond with
 ANALYTICAL LETTERS 2427

the literature. Also, as the literature has shown that SDE-GC/MS is more sensitive for
volatile compound isolation (Khan et al. 2017). This study provided additional informa-
tion through the identification of 50 new volatile compounds in betel leaf varieties for
the first time.
Eugenol was found to be the major volatile compound in all five varieties of betel
leaf, with peak areas ranging from 67.73% to 85.18% (Table 2). Rawat et al. (1989) have
reported the eugenol peak area for betel leaves was from 13.90% to 63.56% using a
hydrodistillation method for extraction. These results further confirm that the developed
SDE-GC/MS method was more sensitive for volatile compounds. Eugenol is widely used
in agricultural applications to protect foods from microorganisms during storage due to
its strong antimicrobial properties (Kamatou, Vermaak, and Viljoen 2012; Sugumaran
et al. 2011). In this study, the eugenol content reported for Misti betel leaves was
between two to three times higher than in the Bangla and Sanchi betel leaves. The
Khasia and BARI Paan 3 betel leaves contained nearly equal concentrations of eugenol.
Previous studies of Indian and Srilankan betel leaves have reported the major volatile
compounds including isoeugenol, (E)-isoeugenol, acetyl eugenol, allylpyrocatechol and
safrole (Pradhan, Biswasroy, and Suri 2014; Arambewela, Kumaratunga, and Dias 2005).
The cultivating environment factor and betel leaf varieties may be responsible for these
differences. Following eugenol, the next most prevalent compounds identified in the betel
leaf varieties were c-muurolene (Bangla and Sanchi), b-caryophyllene (Misti and BARI
Paan 3) and valencene (Khasia). The other major volatile compounds present were euca-
lyptol, linalool, estragole, chavicol, methyl eugenol, a-humulene, eugenyl acetate, d-cadi-
nene, (Z)-a-bisabolene, caryophyllene oxide and humulene epoxide II (Table 2).
Volatile compounds are responsible for the aroma, taste and bioactivity, and are also
employed for chemical fingerprinting. Chemical constituents of different varieties of
plant essential oils are often used for the identification of varieties and the development
of chemotypes. In current study, that betel leaf volatiles were primarily composed of ter-
penoids and phenolics in the same pattern as reported in the literature (Alighiri et al.
2018). Each country and possibly each region may have a distinct betel leaf essential oil,
which may be classified as chemotypes. Chemotypes involving chavicol, germacrene D,
isoeugenol, chavibetol, eugenol, anethole and safrole have been reported for most of the
betel leaf essential oils in the literature (Kumar et al. 2007). This differentiation is pri-
marily due to the ecological and geographical conditions, age of the plant, and harvest
time (Bagamboula, Uyttendaele, and Debevere 2004).
On the basis of chemical contents such as the volatile compounds in plant metrics,
multivariate analysis methods such as principal component analysis (PCA) and hier-
archidical cluster analysis (HCA) have been applied to better understand their classifica-
tion (Chung 1999; Yun et al. 2002). Also, as a classification method, cluster analysis
may be utilized where there are not clear or known classification criteria (Kim et al.
2014). The plant varieties may be classified and separated into different clusters when
they are characterized by principal component analysis, hierarchidical cluster analysis,
partial least square – discriminant analysis, and other multivariate analysis methods
based on the concentrations of these constituents.
Karak et al. (2016) reported that the betel leaf varieties Meetha (Misti) and
Chhaanchi (Sanchi) were distinctly different from the other analyzed varieties of this
2428 M. A. ISLAM ET AL.

species. The results were characterized by principal component analysis and partial least
square – discriminant analysis. The same observations have been demonstrated in this
study. In addition, the Misti and Sanchi betel leaf varieties were grouped in a different
cluster than the other varieties by the use of principal component analysis and hierarch-
idical cluster analysis.

Conclusions
Simultaneous distillation extraction with n-pentane and diethyl ether solvent has been
demonstrated to be suitable for the isolation of the volatile compounds from betel
leaves. SDE-GC/MS provided the determination of 50 volatile compounds for the first
time from betel leaf varieties compared to the reports in the literature. The GC/MS
results demonstrated remarkable differences in the concentrations of volatile compounds
in the betel leaf varieties.
The highest concentration of volatile compounds was found in Misti betel leaves
(13959 mg/kg) while the lowest value was present in Bangla betel leaves (4347 mg/kg).
Eugenol was present at the highest concentration in all varieties with the peak area
varying from 67.73% to 85.18%. The other major volatile compounds present in all vari-
eties of betel leaves were b-caryophyllene, valencene, c-muurolene, chavicol, and caryo-
phyllene oxide.
Through the use of principal component analysis and hierarchidical cluster analysis,
the five varieties of betel leaf were separated and classified into three clusters based on
the volatile compounds determined in this study. The current findings thus further
expand the characterization of the volatile compounds present in betel leaves.

Conflicts of interest
The authors declare no conflicts of interest.

Funding
The authors greatly acknowledge the financial support through the research funds for this study
from Chosun University, Gwangju, Republic of Korea and Bangabandhu Science and Technology
Fellowship Trust, Ministry of Science and Technology, Government of the People’s Republic
of Bangladesh.

ORCID
Kyong Su Kim http://orcid.org/0000-0002-2394-857X

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