Quantification of Zerumbone in Eleven Cultivars of Curcuma longa Using RP-HPLC

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Indian Journal of Pharmaceutical Sciences 1159
November-December 2018
Quantification of Zerumbone in Eleven Accessions of
Curcuma longa Using RP-HPLC
P. R. KSHIRSAGAR, N. V. PAWAR1, S. P. PATIL2, M. S. NIMBALKAR2 AND S. R. PAI3*
Department of Biotechnology, Shivaji University, Vidya Nagar, 1Department of Botany, The New College,
2Department of Botany, Shivaji University, Vidya Nagar, Kolhapur-416 004, 3Amity Institute of Biotechnology
(AIB), Amity University, Mumbai-Pune Expressway, Bhatan, Post Somathne, Panvel, Mumbai-410 206, India
Kshirsagar et al.: Zerumbone from eleven accessions of Curcuma longa
Zerumbone has gained attention in cancer research due to its tumor suppressor potency. Present study
aimed to investigate zerumbone content from eleven cultivars of Curcuma longa by reversed-phase high-
pressure liquid chromatographic analysis. The fresh and dry samples were subjected for the reversed-
phase high-pressure liquid chromatographic analysis of zerumbone including validation studies. Fresh
samples revealed zerumbone content ranged from 0.383-2.179 mg/g while dry samples ranged from 3.209-
8.333 mg/g. Dry samples reected high zerumbone content compared to fresh samples. Among the different
cultivars accessed Selam showed highest content of zerumbone on fresh weight basis while, Alleppey on dry
weight basis. This study provided comprehensive information on zerumbone content in members of family
Zingiberaceae and also quantied it in eleven Curcuma longa cultivars from India.
Key words: Zingiberaceae, Curcuma, RP-HPLC, zerumbone
the edible plant Zingiber zerumbet Smith. It suppresses
various tumours in a potent manner[1]. Rahman et al.[2]
Zerumbone (IUPAC: 2,6,9,9-tetramethyl-[2E,6E,10E]-
cycloundeca-2,6,10-trien-1-one) is a sesquiterpene from

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Indian Journal of Pharmaceutical Sciences 1160
November-December 2018
*Address for correspondence
E-mail: drpaisr@gmail.com
This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
as long as the author is credited and the new creations are licensed under
the identical terms
Accepted 12 October 2018
Revised 04 April 2018
Received 27 March 2017
Indian J Pharm Sci 2018;80(6):1159-1165
has reviewed and comprehensively listed anticancer
properties of zerumbone. It possesses antinociceptive,
antiinammatory, antiulcer, antihyperglycaemic,
immunomodulator and antiplatelet activities[2-4].
Zerumbone has been reported from different species
of Curcuma, Alpinia and Zingiber from the family
Zingiberaceae[2]. A range of variation in contents have
been reported from 0.03 to 84.80 % in the family
(Table 1)[5-26]. It has been admitted that this differences
in zerumbone content in the plants were not due to
geographic or ecological variations but because of their
chemotypes[2,4].
India is the largest producer of turmeric in the world
(93.7 % of the total world production), which is
cultivated in 1 50 000 hectares in India[27,28]. In this
context, there is a need to study and identify high yielding
chemotypes for economically viable genus Curcuma
longa (family: Zingiberaceae) and its cultivars. Use
of turmeric as blood purier, antiseptic and even for
skin conditioning is mentioned in Ayurveda. Also the
plant has been well-documented for its wide range
of pharmacological properties[29,30]. The species is
chiey studied for its phytoconstituents curcumin
and its pharmacological properties[31-33]. Among the
several other constituents present in the plant Gantait
and coworkers[30] reported presence of zerumbone in
C. longa. Apart from this there are no reports of
zerumbone from C. longa (Table 1). However, to the
best of our knowledge, there have been no attempts
to validate and quantify zerumbone content from
C. longa cultivars from India, using reversed-phase
high performance liquid chromatography (RP-HPLC)
analysis.
The study intended to compile various reports
on identication of zerumbone from members of
zingiberaceae and to evaluate zerumbone content
from eleven cultivars of C. longa (viz. Selam, Krishna,
Tekur, Rajapuri, Phuleshwar, Prabha, Bawdhan,
Pachwad, Alleppey, Kuchipudi and Rajampeth) with
its validation using RP-HPLC method.
Eleven Curcuma longa cultivars were obtained from
Indian Council of Agriculture Research (ICAR),
Centre, Sangli, Maharashtra, India, labelled as
CUR-A (Selam), CUR-B (Krishna), CUR-C (Tekur),
CUR-D (Rajapuri), CUR-E (Phuleshwar), CUR-F
(Prabha), CUR-G (Bawdhan), CUR-H (Pachwad),
CUR-I (Alleppey), CUR-J (Kuchipudi), and CUR K
(Rajampeth). The samples were harvested, cleaned
under running tap water to remove soil and dirt, before
bringing it to laboratory.
The rhizomes were air-dried at room temperature
(28±5°) until steady weight was observed. The material
was then ground to ne powder in a laboratory grinder.
One percent extract was prepared by dissolving 1 g
plant powder in 100 ml methanol by keeping it for
24 h. The extracts were ltered, re-volumized and
passed through 0.2 µm nylon lters. The extracts were
stored at –4° until use.
All solvents and chemicals used during the study were
of HPLC grade. HPLC grade zerumbone (≥98.0 %
pure) was obtained from Sigma-Aldrich, Mumbai,
India. An accurately weighed standard zerumbone
was dissolved in known amount of methanol to obtain
mg/ml concentration of stock. The stock solution was
diluted to obtain desired working concentrations (2, 10,
25, 50, 100, 200 µg/ml).
The RP-HPLC analysis was performed on a
chromatographic system consisting of a quaternary
pump, manual injector and dual λ UV absorbance diode
array detector. The built-in chromatographic software
system was used for data processing. Chromatographic
separation was achieved on a Luna, C18 (5 µm)
reversed-phase column (150×4.6 mm, 5 µ). Mobile
phase consisting of acetonitrile, methanol and 0.01 M
potassium dihydrogen orthophosphate (25:55:20) was
used for separation with injection volume 20 µl. The
ow rate was 1 ml/min and the detection wavelength
was set to 254 nm[34]. The analysis time was 10 min for
both standards and samples.
The validation parameters such as accuracy, precision,
limit of detection (LOD), limit of quantication
(LOQ), linearity, range and system suitability test were
assessed by three replicate injections of standards at
appropriate concentrations. The results were subjected
to statistical analysis and are represented as mean±SD
of three independent injections unless mentioned.

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November-December 2018
Indian Journal of Pharmaceutical Sciences
1161
Accuracy of the method was determined by recovery
analysis of the known amounts of standard zerumbone
(2, 10, 50 µg/ml) added to a placebo sample. The
samples were analysed (3 replicates) and the amounts
were calculated from the calibration curve. The
recovery was presented in percent accuracy (calculated
concentration/nominal concentration×100) and it
ranged from 98.60 to 100.63 % indicating acceptable
range as per ICH recommendations for the given
method[35].
Precision of the method was measured according to ICH
recommendations[35]. Three injections of zerumbone at
three different concentrations (2, 10, 50 µg/ml) showed
excellent repeatability (intra-day precision), and
relative standard deviation (RSD) was within 3.76 %.
Inter-day precision was determined by measuring
TABLE 1: DISTRIBUTION OF ZERUMBONE IN MEMBERS OF FAMILY ZINGIBERACEAE
Species Part Content (%) Method Location Reference
Alpinia galangal Rh 44.80 GC-MS Sri lanka [5]
Curcuma amada Rh 00.20 GC-MS India [6]
C. longa Rh NM GC-MS Serbia [7]
SP NM GC-MS China [8]
Rh NM GC-MS Pakistan [9]
Rh NM GC-FID Brazil [10]
C. longa cv ROMA Rh, L NM GC-MS India [11]
C. longa cv Selam Rh 0.22/0.57 RP-HPLC India Present study*
C. longa cv Krishna Rh 0.08/0.45
C. longa cv Tekur Rh 0.15/0.32
C. longa cv Rajapuri Rh 0.13/0.37
C. longa cv Phuleshwar Rh 0.12/0.36
C. longa cv Prabha Rh 0.10/0.45
C. longa cv Bawdhan Rh 0.04/0.64
C. longa cv Pachwad Rh 0.13/0.42
C. longa cv Alleppey Rh 0.16/0.83
C. longa cv Kuchipudi Rh 0.10/0.35
C. longa cv Rajampeth Rh 0.20/0.61
C. purpurascens Rh NM GC-MS Indonesia [12]
C. zedoaria Rh NM GC Vietnam [13]
Xylopia aetiopica Fr 04.00 GC-MS Nigeria [14]
Zingiber aromaticum Rh 17.72 GC-MS Malaysia [15]
Z. cassumunar Rh 01.00 GC-MS India [16]
Z. montanum Rh 02.00 Spec. Bangladesh [17]
Z. ofcinale Rh 36.98 GC-MS Bangladesh [18]
Z. ottensii Rh 25.60 GC-MS Malaysia [19]
Z. spectabile Rh 59.10 GC-MS Malaysia [20]
Z. zerumbet Rh 76.30-84.80 GC-MS India [21]
Rh 01.81 HPTLC India [22]
R 00.16
L 00.09
Fl 00.03
Rh 68.90 GC-MS Malaysia [21]
Rh 00.11 Malaysia [23]
Rh 65.30 GC-MS Tahiti island [24]
Rh 72.30 GC-MS Vietnam [25]
Rh 37.00 GC-MS France [26]
L 00.40 GC-MS France
Rh: Rhizome, R: root; S: stem; L: leaf; Fl: ower; Fr: fruit; SP: sample powder; Spec.: spectroscopy; *content in present study represented
as fresh wt./dry wt. basis

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Indian Journal of Pharmaceutical Sciences 1162
November-December 2018
intra-day variation for triplicated determination of
the same standard zerumbone concentrations (2, 10,
50 µg/ml). The lower RSD values indicated the
acceptable reproducibility of the method.
The LOD and LOQ were calculated based on the
signal:noise ratio. The LOD for zerumbone was
0.04 µg/ml and LOQ was 0.12 µg/ml using above
chromatographic injections. The system suitability
test was performed for the above chromatographic
system used for analysis. This was performed before
sample analysis to pass the system suitability limits.
The evaluation was done using standard zerumbone
200 μg/ml concentration. The capacity factor (k’)
was 0.711 indicating that the zerumbone peak is well
resolved with respect to the void volume. The tailing
factor (T) for zerumbone peak was 1.332, reecting
good peak symmetry. The resolution (Rs) for the
principle peak was 1.026, indicating good separation
of the analytes. The theoretical plate number (n) was
1202.95 for the column used in the study (150×4.6 mm
i.d., particle size 5 μm), thus demonstrating acceptable
column efciency. The RSD of three consecutive
injections of 10 μg/ml concentration of zerumbone
was 0.707 %, indicating fair injection repeatability.
All these results assure the adequacy of the method for
analysis of zerumbone in C. longa.
The linear regression data for the calibration
plots showed a good linear relationship over a six
concentration range from 2 to 200 µg/ml for zerumbone
with respect to the peak areas. It was observed from
the data that, the linearity response of zerumbone is
linear between lower to higher concentration levels
(g. 1 and 2A). Regression Eqn. was obtained using
least-square method and the standard deviation did not
exceed 2 % level.
Six different concentrations (2, 10, 25, 50, 100 and
200 µg/ml) of standard zerumbone were detected
at 254 nm wavelength using RP-HPLC technique.
The analysis yielded proles with a retention time of
5.735±0.28 min (g. 2A). The linearity and sensitivity
of the method was analysed using the set conditions,
three independent calibration curves for the compound
was plotted correlating the detector signals with
concentrations of zerumbone (g. 1). Linear calibration
curves for standards were obtained with coefcient of
determination (R2) not more than 0.999 for standard
zerumbone (g. 1). Results of placebo and blank were
also recorded for the study (g 2B and C).
The applicability of the present method was analysed
using eleven cultivars of C. longa. The extracts obtained
from fresh and dry rhizomes of eleven C. longa cultivars
were subjected to the analysis. The zerumbone content
determined using RP-HPLC method ranged from
0.383±0.019 mg/g (CUR-G) to 2.179±0.109 mg/g
(CUR-A) on fresh weight basis and from 3.209±
0.160 mg/g (CUR-C) to 8.333±0.417 mg/g (CUR-I) on
dry weight basis (g. 2D, E and F). This range was
within the content observed in other species earlier
studied from the same family (Table 1). Members of
genus Curcuma showing lower zerumbone content
compared to Zingiber, Alpinia and Xylopia species
(Table 1). However, the present study showed higher
zerumbone content in different cultivars of C. longa.
Of the eleven-accession evaluated for zerumbone
content, Selam accession (CUR-A) yielded higher
on fresh weight basis whereas, Alleppey accession
(CUR-I) was higher on dry weight basis (g. 3). It
should be noted here that the lowest content on dry
weight basis is equal to or more than the highest
content obtained in fresh weight basis (g. 3). The
percent differences between the two in all the eleven
cultivars were ≥50 %. Accession CUR-G had showed
a higher percent difference of 92 % in content from
fresh and dry weighed extracts followed by CUR-B
(81 %), CUR-I (79 %) and CUR-F (75 %) with ≥70 %
difference.
The content observed in C. longa cultivars in present
study is comparatively less than that of earlier reports
from Z. zerumbet (Table 1). The content observed hereto
in the literature are based on gas chromatography-mass
spectrometry analysis, our result presents for the rst
time a validated RP-HPLC method for quantifying
zerumbone from C. longa, especially different
cultivars. These cultivars represent the agroclimatic
regions of their cultivation in India and can also suggest
its utilization in that respective region. Such study can
y = 33720x + 21144
R² = 0.9995
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
0 50 100 150 200 250
AUC at 254nm
Zerumbone µg/ml
Fig. 1: Calibration curve of standard zerumbone
Six point calibration curve of standard zerumbone at
concentrations of 2, 10, 25, 50, 100 and 200 µg/ml

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Indian Journal of Pharmaceutical Sciences
1163
provide a data to compare use of high yielding C. longa
cultivars to the risk of related diseases.
The study provides comprehensive information on
zerumbone content in members of family Zingiberaceae.
The validated method for quantication of zerumbone
could be used consistently with greater reliability.
Among the C. longa cultivars, Alleppey (CUR-I)
followed by Bawdhan (CUR-G), Rajampeth (CUR-K)
and Selam (CUR-A) had zerumbone content over
0.5 %. This is in the lights of further pharmacological
utility of these cultivars.
A
B
C.
D.
E.
F.
0.0 1.0 2.0 3. 0 4.0 5.0 6.0 7.0 8. 0 9.0 min
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
mAU
254nm4nm (1.00)
/1.548
/2.396
/2.744
/3.470
/3.893
Zerumbone / 10 µg/ml/5. 751
0.0 2.5 5.0 7.5 min
-
40
-
30
-
20
-
10
0
10
mAU
0.0
25.0
50.0
75.0
kgf/cm2
A.Press.(Status)254nm,4nm (1.00)
0.0 1.0 2. 0 3.0 4.0 5.0 6.0 7. 0 8.0 9.0 m in
0
50
100
150
200
250
300
350
mAU
254nm4nm (1.00)
/1.571
/1.986
/2.152
/2.732
/3.768
/4.344
/5.074
CUR-A / Fresh/5.5 27
/6.687
/7.737
/9.634
0.0 1.0 2.0 3. 0 4.0 5.0 6.0 7.0 8.0 9. 0 min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
mAU
254nm,4nm (1.00)
/1.491
/1.883
/2.186
/2.711
/3.784
/4.430
/5.082
CUR-I / Dry/5.5 51
/6.647
/7.781
/9.653
Fig. 2: RP-HPLC proles
(A) Standard zerumbone 10 µg/ml, (B) RP-HPLC prole of blank, (C) RP-HPLC prole of placebo, (D) fresh rhizome sample of
C. longa accession CUR-A, (E) dry sample of C. longa accession CUR-A and (F) purity curve of sample C. longa accession CUR-I

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Acknowledgements:
Authors thank the Director, AIB, Amity University,
Mumbai, HOD, Biotechnology and HOD, Botany,
Shivaji University, Kolhapur for their support. Thanks
are also due to Dr Jaykumar Chavan, Department
of Botany, YCIS, Satara, India for gifting standard
zerumbone. NVP is indebted to the Principal and HOD,
Department of Botany, The New College, Kolhapur.
Conict of interest:
There is no conict of interest, nancial or otherwise
associated with this project.
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CUR
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