Assessment of phytochemical diversity in essential oil composition of eighteen Piper nigrum (L.) accessions from southern India

Abstract

The chemical composition of essential oils isolated from dried berries of eighteen accessions of black pepper (Piper nigrum L.) from southern India was evaluated by gas chromatography-mass spectrometry analyser. The volatile oil content extracted by hydrodistillation method was ranged from 2.30% to 5.00% (w/w) among the accessions. In total, 41 constituents accounting about 98.3-100% of the total essential oil composition were identified. The main fractions were monoterpene hydrocarbons (60.6-81.2%) followed by sesquiterpene hydrocarbons (16.6-31.1%), oxygenated sesquiterpenes (0.2-3.4%) and oxygenated monoterpenes (0.2-0.9%). The highest content of sabinene and (E)-β-caryophyllene was obtained from the accessions Irumbirakki and ACC53, respectively. The present study revealed the existence of a new essential oils/chemotypes (safrole and β-ylangene) of the black pepper was not described before from southern India. The major constituents of black pepper essential oil (BPEO) can be utilized in the food, perfumery, aroma and pharmaceutical industries.

Full text

Assessment of phytochemical diversity in essential oil composition of eighteen
Piper nigrum (L.) accessions from southern India
K. Ashokkumar
a
, S. Vellaikumar
b
, M. Murugan
a
, M.K. Dhanya
a
, A. Karthikeyan
c
, M. Akilan
b
,
G. Ariharasutharsan
b
, M. Nimisha
a
and S. Aiswarya
a
a
Cardamom Research Station, Kerala Agricultural University, Idukki, Kerala, India;
b
Agricultural College and Research Institute, Tamil Nadu
Agricultural University, Madurai, Tamil Nadu, India;
c
Subtropical Horticulture Research Institute, Jeju National University, Jeju, Republic of
Korea
ABSTRACT
The chemical composition of essential oils isolated from dried berries of eighteen accessions of
black pepper (Piper nigrum L.) from southern India was evaluated by gas chromatography-mass
spectrometry analyser. The volatile oil content extracted by hydrodistillation method was ranged
from 2.30% to 5.00% (w/w) among the accessions. In total, 41 constituents accounting about 98.3–
100% of the total essential oil composition were identied. The main fractions were monoterpene
hydrocarbons (60.6–81.2%) followed by sesquiterpene hydrocarbons (16.6–31.1%), oxygenated
sesquiterpenes (0.2–3.4%) and oxygenated monoterpenes (0.2–0.9%). The highest content of
sabinene and (E)-β-caryophyllene was obtained from the accessions Irumbirakki and ACC53,
respectively. The present study revealed the existence of a new essential oils/chemotypes (safrole
and β-ylangene) of the black pepper was not described before from southern India. The major
constituents of black pepper essential oil (BPEO) can be utilized in the food, perfumery, aroma and
pharmaceutical industries.
ARTICLE HISTORY
Received 29 January 2020
Accepted 27 August 2021
KEYWORDS
Piper nigrum l; essential oil;
sabinene; 3-carene; (E)-β-
caryophyllene; cluster
analysis
1. Introduction
Black pepper (Piper nigrum L.) belongs to the
Piperaceae family, is extensively used as condiment
worldwide, and is native to Kerala state in southern
India (1). It is widely cultivated in Vietnam, India,
Malaysia, Indonesia, Brazil, China, Sri Lanka and
Thailand (2). In most of the Asian countries, black
pepper is used as a major spicy ingredient for food
preparations, and it also has potential application in
folk medicine, perfumery, and insecticides (3). The use
of this plant as a source of some natural products has
great interest by several researchers worldwide. Black
pepper extract and black pepper essential oil (BPEO)
have several potential biological activities and health
benefits. Black pepper has been used traditionally to
treat cough, cold, dyspnea, throat disorders, intermit-
tent fever, dysentery, stomachache, worms and piles (4).
The plant is also used as an anti-inflammatory, anti-
pyretic, and treatment for epilepsy and snakebite (5–7).
Besides traditional medicinal usages, potential modern
medicine applications by various investigations justified
its antimicrobial, anticancer, antidiarrhoeal, antidepres-
sant, antiobesity and anthelmintic activities (8–14).
Black pepper is recognised as the ‘king of spices’ due
to its possible health benefits.
Black pepper berries contain terpenes, flavonoids,
carotenoids and other bioactive compounds (15). The
major bioactive constituents present in BPEO are
mainly responsible for its distinct flavour and aromatic
smell (16). The essential oil yield from the black pepper
dried berries varied from 1.2% to 5.1% when estimated
by the hydrodistillation extraction method (17,18). The
composition of BPEO could depend upon the varieties,
origin of sample, location and season of the harvested
sample (19–21). BPEO is rich in monoterpene constitu-
ents: sabinene (0.1–27.5%), limonene (15.1–20.8%),
3-carene (9.2–32.6%), α-phellandrene (0.1–7.4-%), α-
pinene (3.88–6.48%) and α-thujene (0.6–2.9%) (20–
22). Presence of sesquiterpene components, such as
(E)-β-caryophyllene (9.5–27.0%), β-bisabolene (1.3–
8.0%), α-humulene (1.1–2.4%) and α-copaene (0.2–
5.5%), in BPEO was reported by Sruthi et al. (21).
BPEO bioactive constituent nerolidol and (E)-β-
caryophyllene exhibited insecticidal and anesthetic
effects, respectively (23–25).
Several studies revealed that huge variability has
occurred in the essential oil extracted from dried
black pepper seeds; however, the origin of most of
CONTACT K. Ashokkumar biotech.ashok@gmail.com
JOURNAL OF ESSENTIAL OIL RESEARCH
https://doi.org/10.1080/10412905.2021.1975578
© 2021 Informa UK Limited, trading as Taylor & Francis Group

these samples is not sufficiently defined. They are
probably commercial samples obtained from markets
with unknown original habitats or maybe bulk samples
constituted by mixtures of various black pepper vari-
eties, which could not represent the individual chemo-
types. Moreover, the proportion of predominant
constituents of BPEO could be affected by various
factors like varieties, origin, soil type, season, storage
conditions and extraction methods. Therefore, most of
the published results were not scientifically concluded
with the real chemical diversity of the BPEO. In this
context, the current study’s objective was to describe
the essential oil contents of eighteen prospective
P. nigrum accessions based on consumer and manu-
facturer demand.
2. Materials and methods
2.1. Plant material
Eighteen promising black pepper accessions were col-
lected from experimental plots at Cardamom Research
Station, Pampadumpara, Kerala, India, located between
coordinate’s 9
°
45ʹ N latitude, 77
°
10ʹ E longitude and
altitude is 1100 m above mean sea level. Plants were
grown at the uniform field conditions were selected to
avoid the impact on the environment. The matured
berries were harvested during April–May 2019 and
sun dried to evaluate essential oil content and its com-
position. Sufficient quantities (~100 g) of dried berries
of each accession were stored at room temperature.
2.2. Extraction of essential oils
The dried berries of each black pepper accession were
ground into fine powder. Twenty grams of powdered
sample was placed in a 500-ml distillation flask.
A hydrodistillation runtime of 3 h was used to obtain
optimum yield without drastically affecting the oil con-
stituents (26). Oil yield was estimated with an average of
three replications. A Dean–Stark distillation apparatus
determined the moisture content of all the black pepper
accessions (27). Essential oil content was calculated on
dry weight basis through the following formula: Dry
weight basis (%) = [Volatile oil yield [%]/(100 – moist-
ure (%)] × 100. The essential oil was carefully collected
and dried over anhydrous sodium sulphate and 100 µl of
the oil diluted with 1.5 ml of hexane (28). A diluted
sample was stored in a sealed glass vial for GC-MS/FID
analysis.
2.3. Analysis of essential oils
The quantitative analysis of essential oil was carried out
through GC-MS/FID equipment (QP2010 Ultra,
Shimadzu Corporation, Kyoto, Japan). GC was
equipped with a fused silica capillary column, Rxi-5 Sil
MS (20 m, 0.18 mm ID), with a film thickness of
0.18 μm. Samples (50 µl) were injected with a split
ratio of 1:100, and helium gas (99.99%) flow rate was
constantly maintained at 1 ml/min (28). Triplicate ana-
lyses were carried out for all the essential oil samples.
The oven temperature was programmed at 70°C for
15 min, and then gradually increased 6°C/min to
200°C and then 30°C/min to 280°C (10 min hold).
A detector splitting system simultaneously acquired
the MS and FID data with a 4:1 (MS: FID) split flow
ratio. MS conditions were electron energy 70 eV, elec-
tron impact (EI) ion source temperature is 250°C, and
transmission line temperature 280°C. The MS data
(TIC, total ion chromatogram) were acquired full scan
mode [mass scan range (m/z) of 50–650 amu] at a scan
rate of 0.3 scan/s. The injector and FID detector tem-
perature was 250°C. The gas hydrogen (30 ml/min), air
(300 ml/min), and helium (30 ml/min) were supplied
for FID. Each constituent was quantified through FID
peak area normalization (%).
2.4. Identication of essential oil constituents
The components of BPEOs were identified by compar-
ing based on linear indices relative to series of n-alkanes
(C8–C20) at the same chromatographic conditions and
by comparison of retention indices with those present in
NIST and Wiley libraries as well as with literature data
(29). Identification of certain compounds [sabinene,
3-carene, limonene, α-pinene, β-phellandrene, α-
phellandrene and (E)-β-caryophyllene] was further con-
firmed by co-injection of their authentic standards
(Sigma-Aldrich, Mumbai, India) under the same chro-
matographic conditions mentioned above. The indivi-
dual components concentration (%) of oil is expressed
as a percent peak relative to the total area of essential oil
by electronic integration without correction factors.
2.5. Statistical analysis
The results reported in this study are mean values of at
least three technical replications for three biological
replications (n = 9) unless otherwise stated. Statistical
analysis was conducted to study the significant effects of
2K. ASHOKKUMAR ET AL.

essential oil yield of black pepper accessions using ana-
lysis of variance (ANOVA) and Duncan’s multiple
range test (DMRT) at the 0.05 level of significance.
The analytical data of the chemical compounds of the
essential oils were subjected to two multivariate analyses
(agglomerative hierarchical cluster analysis [AHC] and
principal component analysis [PCA]) using the Excel
program plug-in XLSTAT version 2020.3.1. The AHC
was used to understand the relationship between the
populations based on the essential oil compositions
and determine their chemotypes (30,31). Euclidean dis-
tance was selected to measure the dissimilarity, and the
unweighted pair group average method was used for
cluster description. GraphPad Prism software version
9.2. was used to obtain the histograms that showed the
major compounds, with the respective standard errors
of the means for each cluster identified in the cluster
analysis.
3. Results and discussion
The yield of essential oil from dried berries of eighteen
selected black pepper accessions by hydro-distillation
extraction method was varied from 2.3% to 5.0%
(Table 1). The highest essential oil content was observed
in accession Aryanmundi (BP2: 5.0%), followed by the
local most popular cultivar Karimunda (BP4: 4.5%), was
within the range of essential oil reported in Karimunda
(4.1–4.6%) for three consecutive seasons by Menon et al.
(32). According to the authors, the least oil content
(1.7–2.2%) was in accession Kalluvally, which was con-
firmed in the present study results also showed the least
oil content is 2.3% in accession ACC 53 (BP11). The
volatile oil content in spice crops usually varied with
various extraction methods, varieties, growing seasons,
soil types and locations (17, 18, 28, 32, and 32).
The essential oil characterization of eighteen acces-
sions of P. nigrum revealed forty-one constituents. The
relative percentage of volatile constituents identified in
the essential oil of P. nigrum is summarized in Table 1.
Previous studies reported that predominant constitu-
ents of essential oil during chemical analysis are sabi-
nene, 3-carene, D-limonene, α-pinene, β-pinene, (E)-β-
caryophyllene, β-phellandrene, α-phellandrene, β-
bisabolene, α-
copaene and elemol (19–21,25,32–34) and were con-
firmed with the present study.
The chemical profile of essential oil from the eighteen
accessions showed forty-one constituents, which com-
prised about 98.3–100%. Among them, monoterpene
hydrocarbons dominated in the oil composition with
60.6–81.2% followed by sesquiterpene hydrocarbons
(16.6–31.1%), phenylpropene (4.1%), oxygenated
sesquiterpenes (0.2–3.4%), and oxygenated monoter-
penes (0.2–0.9%), (Table 1). Sabinene (11.2–41.6%),
D-limonene (16.7–24.2%) and α-pinene (4.6–16.1%)
were the major monoterpenes hydrocarbon constituents
present in the BPEO of all the accessions. However,
3-carene (0.2–24.9%) is major in sixteen accessions.
The other monoterpenes present in substantial amounts
were β-phellandrene (0.2–7.1%) α-phellandrene (0.3–
6.1%), and α-thujene (0.7–4.7%). The highest sabinene
content of 41.6% was noted in the accession Irumbirakki
(BP3), which was about twofold higher as compared to
the previous report of black pepper variety Panniyur 3
(BP7: 17.2%) (35). However, this high level observed
might be due to a change in the variety, soil type, and
location. Among the accessions, ACC 53 (BP11) showed
a higher monoterpene constituent of α-pinene (16.1%).
The accession Panniyur 4 (BP8) had a greater level of β-
pinene (14.2%) and D-limonene (24.2%), whereas the
accession Karimunda (BP4) was richer in 3-carene
(25.0%) and α- phellandrene (6.1%).
In the present study, twenty-three sesquiterpene con-
stituents were identified, among them (E)-β-
caryophyllene (10.5–18.9%) is the predominant consti-
tuent in all the evaluated black pepper accessions. The
content of four sesquiterpene, hedycaryol (0.2–11.3%),
ß-bisabolene (0.2–6.2%), α-selinene (0.3–5.3%), α-
copaene (0.2–4.6%), and germacrene D (0.2–2.6%) var-
ied substantially among accessions. The accession ACC
53 (BP11) had higher (E)-caryophyllene (18.9%) and is
within the range of earlier published results for black
pepper accession Ottaplackal (15.5–21.7%) evaluated
for three consecutive seasons (34).
Furthermore, the higher amount of β-bisabolene
(6.2%), α-selinene (5.3%), α-copane (4.6%) and γ-
elemene (3.1%), is present in accessions ACC 57,
Vellakari, Panniyur 3 and Vellamundi, respectively
(Table 1). In this study, we have also identified two
phenylpropene constituents, myristicin (3.8%) and
safrole (0.4%), in the accession ACC 106 (BP13),
which makes it unique from other accessions evaluated.
Several chemotypes such as sabinene, 3-carene,
D-limonene, α-pinene, (E)-β-caryophyllene, β-
phellandrene, α-phellandrene, α-thujene, β-bisabolene,
α-
selinene and α-copaene have been reported in black
pepper (19–21,25,33).
The principal component analysis (PCA) was per-
formed with BPEO chemical constituents. Results
showed large variability in the chemical composition
of essential oils with cumulative variance. The PCA
constructed the first two principal axes (F1 and F2)
displayed 93.39% of the total variance (Figure 1). Also,
axes F1 and F2 account for 86.07% and 7.32% of the
JOURNAL OF ESSENTIAL OIL RESEARCH 3

Table 1. Essential oil composition in dried berries of eighteen accessions of Piper nigrum.
No. Compound name RI
a
RI
b
Area %
BP1 BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 BP11 BP12 BP13 BP14 BP15 BP16 BP17 BP18
C1 α-Thujene 924 930 1.3 2.1 4.7 0.7 1.4 1.9 2.2 2.1 2.3 3.0 1.8 2.1 1.4 3.4 1.9 3.1 1.4 2.5
C2 α-Pinene 948 943 7.5 7.1 6.4 6.4 4.6 6.0 6.7 7.4 5.9 7.5 16.1 6.6 6.4 7.5 5.6 9.8 8.7 5.9
C3 Camphene 951 954 - - - - - - 0.1 - - - 0.5 - 0.2 0.1 - 0.2 - 0.1
C4 Sabinene 969 979 11.2 17.4 41.6 - 12.1 18.2 18.9 20.2 17.0 24.0 16.1 19.1 13.8 28.6 22.8 25.4 13.2 19.3
C5 β-Pinene 988 990 7.8 9.9 6.8 13.2 8.7 12.7 12.9 14.2 8.6 12.6 11.1 12.9 12.4 10.6 11.4 10.7 11.7 9.8
C6 β-Myrcene 988 990 1.4 1.9 1.1 2.3 1.9 1.8 1.8 1.5 2.1 1.9 1.8 1.6 2.1 1.8 1.3 1.8 2.2 2.0
C7 α-Phellandrene 1002 1002 2.8 3.7 0.8 6.1 3.3 0.9 0.4 0.5 4.4 0.6 1.1 0.6 2.8 0.5 0.3 0.5 4.6 3.0
C8 3-Carene 1008 1011 11.1 13.5 9.0 24.9 10.7 3.2 0.2 0.8 13.5 0.6 0.9 1.2 9.9 0.6 1.0 0.3 15.4 12.0
C9 β-Cymene 1020 1024 0.4 0.4 - 0.3 0.3 0.3 - - 0.4 - 0.2 - 0.2 0.2 0.3 - - 0.8
C10 Limonene 1024 1029 16.7 17.6 - 22.2 19.7 24.1 23.9 24.2 16.7 22.5 19.7 23.7 23.4 21.0 20.0 20.1 21.3 19.1
C11 β-Phellandrene 1025 1029 - 2.9 7.1 - 0.2 - - - 2.9 3.3 - - - 4.3 3.0 3.7 - 2.5
C12 β-Ocimene 1030 1030 - 0.1 0.7 - - - - - 0.4 0.6 0.2 0.2 0.3 0.2 - 0.4 0.3 1.0
C13 α-Terpinene 1052 1059 0.5 0.6 0.2 1.1 0.6 0.6 0.5 0.3 1.2 - 0.3 0.2 0.7 0.6 - - 1.3 -
C14 β-Linalool 1082 1095 0.4 0.3 0.3 - 0.8 0.3 - 0.5 - 0.5 - - 0.2 - - 0.4 -
C15 Terpinen-4-ol 1174 1177 - 0.2 0.4 - - - 0.2 - 0.2 - 0.4 0.2 - 0.2 0.3 0.3 - -
C16 α-Terpinyl acetate 1333 1300 - - - - - - - - - - - - - - 0.4 - 1.2 0.5
C17 Safrole 1327 1327 - - - - - - - - - - - - 0.4 - - - - -
C18. p-Menth-3-ene 1337 1344 - 0.7 0.6 0.3 - 0.4 0.5 0.5 0.4 1.0 0.5 0.6 0.9 - - 0.6 1.3 -
C1 α-Cubebene 1344 1351 - - - - - 0.2 0.2 - - - 0.2 - - - - - -
C20 α-Copaene 1374 1376 4.3 - 0.5 - 3.6 4.0 4.6 4.0 0.2 1.4 0.2 4.1 1.0 - 4.0 2.4 - 0.2
C21 β-Ylangene 1419 1419 - - - - - - - - - - - - - - 0.7 0.5 - -
C22 α-Bergamotene 1430 1434 - - - - - - - - - 0.6 - - - - - - - -
C23 γ-Elemene 1434 1436 - - 0.9 - - - - - - - - - - - - - 0.6 3.1
C24 α-Guaiene 1437 1439 - - - - - - - - 0.4 - 0.5 - - - - - - 1.1
C25 (E)-β-Famesene 1440 1440 - - - - 0.3 0.3 0.3 - - 0.4 - 0.3 - - - - - 0.3
C26 Humulene 1452 1454 0.9 0.9 0.9 1.4 1.1 1.0 0.9 0.9 1.2 0.7 1.4 0.8 0.9 1.0 0.9 0.7 0.8 0.8
C27 (E)-β-Caryophyllene 1464 1466 14.0 15.7 16.0 11.5 14.8 13.2 14.4 16.5 13.6 10.5 18.9 13.0 13.3 16.6 14.7 11.9 11.4 12.5
C28 Germacrene D 1480 1483 1.0 2.6 0.9 - 1.2 0.8 0.3 0.8 - - 1.4 1.0 0.4 - 0.2 0.4 0.4 0.9
C29 Muurolene 1500 1500 - - - - 1.2 - - - - - - - - - 1.2 - -
C30 α-Cuprenene 1505 1505 - - - - - 0.5 0.2 - - 0.5 - 0.2 - 0.1 - - - -
C31 α-Bulnesene 1509 1509 - 0.4 - 3.3 - - - - 1.1 0.4 3.2 - - - - - - -
C32 Myristicin 1516 1516 - - - - - - - - - - - - 3.8 - - - - -
C33 α-Selinene 1520 1520 0.3 0.7 - 4.7 1.1 3.9 1.7 0.3 5.3 1.9 1.9 3.5 1.6 0.5 2.4 0.4 0.9 -
C34 Cadinene 1521 1522 - - - - - 2.0 2.5 - - 1.1 - - - - - 1.3 - -
C35 Bisabolene 1529 1531 3.2 0.2 - - 2.4 3.3 5.4 3.3 - 3.8 0.9 6.2 2.3 1.0 4.8 2.1 1.3 1.4
C36 Elemol 1548 1549 11.3 - - - 5.5 - - 0.2 - 0.6 - - - - - 2.2 - -
C37 Nerolidol 1564 1563 1.0 - - - 1.5 - - - 0.9 - - - - - - - - 1.3
C38 Caryophyllene oxide 1582 1583 - - - - - 0.3 0.2 - - - - - - - 1.1 - 0.3 -
C39 Spathulenol 1577 1578 - - - - - - - - - - - - - - 0.3 - - -
C40 Cederol 1600 1600 1.5 - - - 1.4 - 0.7 0.8 - - - 0.9 1.1 - 0.6 - - -
C41 p-Cresol 1668 1674 - - - - - - - - - - - - - 0.7 2.0 - - -
Monoterpene hydrocarbons 60.6 77.2 78.3 77.2 63.5 69.7 67.5 71.0 75.4 76.6 69.8 68.1 73.7 79.4 67.9 75.9 81.2 78.4
Oxygenated monoterpenes 0.4 0.5 0.7 - 0.8 0.3 0.2 - 0.7 - 0.9 0.2 - 0.4 0.3 0.3 0.4 -
Total monoterpene 61.0 77.7 79.0 77.2 64.3 70.0 67.7 71.0 76.1 76.6 70.7 68.3 73.7 79.8 68.2 76.2 81.6 78.4
Sesquiterpene hydrocarbons 23.6 21.3 19.8 21.3 25.5 29.5 31.1 26.3 22.1 22.1 28.8 29.8 20.4 19.2 27.7 21.4 16.6 20.3
Oxygenated sesquiterpenes 1.0 - - - 1.5 0.3 0.2 - 0.9 - - - - 0.7 3.4 - 0.3 1.3
Total sesquiterpene 24.6 21.3 19.8 21.3 27.0 29.8 31.3 26.3 23.0 22.1 28.8 29.8 20.4 19.9 31.1 21.4 16.9 21.6
Total phenylpropene - - - - - - - - - - - - 4.1 - - - - -
(Continued)
4K. ASHOKKUMAR ET AL.

Table 1. (Continued).
No. Compound name RI
a
RI
b
Area %
BP1 BP2 BP3 BP4 BP5 BP6 BP7 BP8 BP9 BP10 BP11 BP12 BP13 BP14 BP15 BP16 BP17 BP18
Alcohol + Phenol 12.8 - - - 7.0 - 0.7 1.0 - 0.6 - 0.9 1.1 - 0.6 2.2 - -
Total compounds (%) 98.4 99.0 98.8 98.5 98.3 99.8 99.7 98.3 99.1 99.3 99.5 99.0 99.3 99.7 99.9 99.8 98.5 100
Essential oil (%) 4.0
c
5.0
a
3.5
d
4.5
b
2.5
g
2.5
g
3.0
f
3.0
f
3.5
d
3.5
d
2.3
h
3.5
d
3.1
e
3.5
d
2.5
g
4.0
c
4.0
c
2.5
g
The means of essential oil content were statistically analyzed using ANOVA. Within columns, means followed by the same letter (a–h) are not significantly different according to Duncan’s multiple range test (p < 0.05). (-), not
detected,
a
RI = Retention index (experimental) on Rxi5 Sil MS column;
b
RI = Retention index from literature (Adams 2017); Explanation of code of black pepper accessions, BP1: Thevanmundi; BP2: Aryanmundi; BP3:
Irumbirakki; BP4: Karimunda; BP5: Panniyur1; BP6: Panniyur2; BP7: Pannniyur3; BP8: Panniyur4; BP9: Vellakari; BP10: ACC33; BP11: ACC53; BP12: ACC57; BP13: ACC106; BP14: HP39; BP15: HP20052; BP16: PRS88; BP17:
C-1090; BP18: Vellamundi.
JOURNAL OF ESSENTIAL OIL RESEARCH 5

total variance, respectively. The first axes accounted
positive correlation with major essential oil constitu-
ents, α-pinene (r = 0.81), sabinene (r = 0.90), β-pinene
(r = 0.96), 3-carene (r = 0.90), D-limonene (r = 0.95) and
(E)-β-caryophyllene (r = 0.97). The second axis
accounted for the positive correlation of β-pinene, β-
myrcene, α-phellandrene, 3-carene, D-limonene, α-
terpinene, (E)-β-caryophyllene, α-bulnesene, α-
selinene and elemol, and other constituents were nega-
tively correlated.
Hierarchical clustering analysis was carried out to
understand the relationship among the eighteen black
pepper accessions based on the EO compounds using
Euclidean distance assessing divergence between the
samples (30). A dendrogram obtained by cluster analy-
sis performed on the EO composition of eighteen black
pepper accession showed five main clusters (Figure 2).
Cluster 1 and cluster 3 are made up of eight and seven
accessions, respectively. Cluster 2, cluster 4, and cluster
5 each composed one accession. Cluster 1 formed two
sub-clusters namely, PRS 88, ACC 33, HP20052 and HP
39; ACC57, Panniyur 3, Panniyur 2 and Panniyur 4
while cluster 3 formed 3 sub-clusters, namely C1090
and ACC106; Vellakari, Aryamundi and Vellamundi;
Panniyur 1 and Thevanmundi. However, the accession
ACC 53 and Karimunda and Irumbirakki composed
clusters 2, 4 and 5, respectively. Accessions belonging
to the same group show more similarity in the essential
oil composition. In brief, sabinene, 3-carene,
D-limonene, α-pinene, β-pinene, (E)-β-caryophyllene,
β-phellandrene, α-phellandrene, β-bisabolene, α-
copaene and elemol were found in higher concentra-
tions in BPEOs and formed five chemical clusters
(Figure 3). The accessions under cluster 1 had higher
D-limonene & bisabolene, and cluster 3 grouped acces-
sions had higher in elemol content. The accession ACC
53 (BP11) had the highest (E)-β-caryophyllene (18.9%)
and α-pinene (16.1%) in cluster 2, while the accession
Karimunda had the highest 3-carene (24.9%) in cluster
4. Furthermore, the accession Irumbiraki had highest
sabinene (41.6%) in cluster 5.
The existence of (E)-β-caryophyllene in the black
pepper accession ACC 53 of cluster 2 suggests an anti-
fungal activity (36). Also, several previous studies sug-
gest that BPEO has antimicrobial, anti-inflammatory,
and antioxidant activity due to the presence of major
essential oil constituents like sabinene, limonene, and α-
pinene (33,37–39). The knowledge of the chemo-
Figure 1. Distribution of the chemical compounds of the essential oils from eighteen Piper nigrum accessions in relation to the two
major compounds by the principal component analysis (PCA). The details of chemical constituents: C1–C41 from Table 1.
6K. ASHOKKUMAR ET AL.

variability of BPEO can pave the way to promote studies
that investigate the therapeutic potential of the species.
These findings could be useful for researchers to choose
particular accessions for developing varieties with
higher essential oil constituents and high yield in black
pepper.
4. Conclusion
The present study provided extensive data on the var-
iation of essential oil content and its composition from
eighteen promising black pepper accessions of south
India. A significant variation was observed in the dis-
tribution of various constituents in the studied black
pepper accessions. All the accessions were grown
under the same environmental condition; therefore,
the effect of ecological factors such as soil type,
shade, and location, etc., can be excluded from com-
positional variation in BPEO. Characterization of
BPEO composition from different accessions could be
useful for further studies, including pharmacological
and pesticide activity. This study has identified that the
essential oil of black pepper accessions predominantly
consists of monoterpene constituents followed by ses-
quiterpenes and phenylpropenes. This study also
observed that accession Irumbirakki, Panniyur 4 and
Karimunda had the highest sabinene, D-limonene, and
3-carene, respectively, compared to other accessions.
Furthermore, the accession ACC 53 (BP11) had the
highest α-pinene (16.1%) and (E)-β-caryophyllene
(18.9%) content, whereas ACC 57 (BP12) had
a greater level of β-bisabolene (6.2%). These findings
indicated a high potential for domestication, cultiva-
tion, and selective breeding. These findings could also
be used as a database for the black pepper commerce
and pharmaceutical industries. Farmers will also ben-
efit from the study’s findings by adopting high-quality
cultivars. Future investigations using more promising
black pepper germplasms/accessions will help identify
the novel bioactive constituents that are mandatory for
the pharmaceutical, flavor, and perfume industries.
Clear idea about medicinally important components
of essential oil against various diseases by systematic
clinical studies will expand the medical application of
BPEO.
Acknowledgments
The authors express their gratefulness to the AICRP on Spices,
Indian Council of Agricultural Research, New Delhi, India.
Figure 2. Dendrogram obtained by agglomerative hierarchical cluster analysis of the essential oil constituents of the eighteen
accessions of Piper nigurm under study based on unweighted pair group average method using the Euclidean distances.
JOURNAL OF ESSENTIAL OIL RESEARCH 7

Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
K. Ashokkumar http://orcid.org/0000-0002-0440-6310
A. Karthikeyan http://orcid.org/0000-0002-6270-5597
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