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Short Communications

HPTLC Analysis of Myristicin and Safrole in Seed Powder


of Myristica fragrans Houtt.
Vidya V. Dighe and Gauri A. Charegaonkar*

Key Words
HPTLC
Myristicin
Safrole
Myristica fragrans Houtt.
Quantification

1 Introduction
The dried ripe seed of the evergreen aromatic tree Myristica
fragrans Houtt. (Fam. Myristicaceae), commonly called nutmeg, is cultivated in many tropical countries, for example India,
Sri Lanka, Grenada, Indonesia, and Malaysia [1, 2]. The seeds
have been used as a stimulant, carminative, narcotic, emmenagogue, and abortifacient [1], although their primary use is as a
spice [3].
The seeds contain a large variety of organic compounds; the
most abundant are myristicin or methoxysafrole (4-methoxy-6(2-propenyl)-1,3-benzodioxole) then safrole (5-(2-propenyl)-3benzodioxole) [4].
Nutmeg is abused because myristicin has psychotropic and hallucinogenic properties [5]. Human studies indicate that myristicin at a dose of 67 mg kg1 body weight can have psychopharmacological effects [1]. Safrole has been reported to be a
possible carcinogen [6] and a study has revealed that safrole is
capable of altering the DNA in human hepatoma (HepG2) cells
and may thus contribute to human carcinogenesis [7]. Because
of the harmful effects of myristicin and safrole, a need was felt
to simultaneously quantify them in dried seed powder of
M. fragrans from different regions.
A literature survey reveals that an HPLC method has been
reported for analysis of myristicin and safrole in nutmeg [4].
Although a TLC method for identification of myristicin from
aril and seed powder of M. fragrans [8], and a densitometric
TLC method for quantification of myristicin in seeds of
Anethum graveolens and Carum carvi have been reported [9],
no HPTLC method has been reported for simultaneous quantification of myristicin and safrole in crude seed powder of M. fragrans. In this research work, a simple and precise method has
been established for simultaneous quantification of myristicin
and safrole in the seed powder of M. fragrans.

V.V. Dighe and G.A. Charegaonkar, S.P. Mandalis Ramnarain Ruia College,
Matunga, Mumbai 400 019, India.
E-mail: gauricharegaonkar@gmail.com

Journal of Planar Chromatography 22 (2009) 6, 445448


0933-4173/$ 20.00 Akadmiai Kiad, Budapest

2 Experimental
2.1 Chemicals, Reagents, Materials, and Solutions

Methanol and toluene (E. Merck, Mumbai, India) were of analytical grade. Myristicin standard (purity 98.3%) was from
SigmaAldrich Chemie (Germany). Safrole standard (purity
97%) was a gift from Drug Testing Laboratory (DTL), Jaipur,
India.
Stock solutions (0.5 mg mL1) each of myristicin and safrole
were prepared in methanol. Working standard solutions
(0.05 mg mL1) were prepared from the stock solutions.
Seeds of Myristica fragrans Houtt. were obtained from three
locations (Konkan region in India, Sri Lanka, and Grenada). The
sample from Konkan (India) was authenticated by the National
Botanical Research Institute (NBRI), Council of Scientific and
Industrial Research, Lucknow, India (voucher specimen no:
94556). The other samples were compared with the authenticated sample and their authenticity was established.
2.2 Sample Preparation

The whole seeds of M. fragrans were powdered and the powder


was passed through a BSS no. 85-mesh sieve. The powdered
samples were stored in separate air-tight container at room temperature (28 2C).
Methanol (10 mL) was added to accurately weighed seed powder
(approx. 1 g) and the mixture was sonicated for 45 min. Each
extract solution was then filtered. For analysis of myristicin from
each sample solution, 1 mL filtrate was diluted to 2 mL with
methanol and this was used as the corresponding sample solution.
2.3 Chromatography

Chromatography was performed on 20 cm 10 cm aluminum


backed HPTLC plates coated with 0.2-mm layers of silica gel 60
F254 (Merck, Mumbai, India). Before use, the plates were prewashed with methanol and activated in an oven at 110C for
10 min. Standards and samples were applied as sharp bands
DOI: 10.1556/JPC.22.2009.6.11

445

Peak area

Residuals

Short Communications

Concentration (ng/band)
Figure 3
Plot of residuals for myristicin in the linear working range.

Figure 1
Calibration plot for myristicin in the linear working range.

Residuals

Concentration (ng/band)

Concentration (ng/band)
Peak area

Figure 4
Plot of residuals for safrole in the linear working range.

Table 1
Results from method validation.

Concentration (ng/band)
Figure 2
Calibration graph for safrole in the linear working range.

6 mm long, 8 mm from bottom of the plate and 15 mm from


the edge, by means of a CAMAG (Muttenz, Switzerland)
Automatic TLC Sampler 4 (ATS 4). The application speed was
150 nL s1. Plates were developed to a distance of 70 mm, with
toluene as mobile phase, in a CAMAG Automatic Developing
Chamber 2 (ADC 2). Before development the plates were saturated with mobile phase vapor for 20 min. After development
they were dried in ambient air for 5 min. The plates were then
scanned at 210 nm for myristicin and 290 nm for safrole, in
absorbancereflectance mode, by means of TLC Scanner 3
(CAMAG) with WinCATS software, version 1.4.4, using the
deuterium lamp. Photodocumentation of the plates was by use
of a TLC Visualizer (CAMAG).
2.4 Method Validation

The method was validated in accordance with ICH guidelines.


Calibration plots for myristicin and safrole were obtained by
plotting peak area against amounts of myristicin and safrole
applied to the plates. The adequacy of the regression model was
examined by plotting the residuals for the calibration plots. The
limits of detection (LOD) and quantitation (LOQ) were defined
as the amounts for which the signal-to-noise ratios were 3:1 and
10:1, respectively. Instrumental precision was studied by repeated analysis (n = 10) of myristicin standard (500 ng per band) and
safrole standard (400 ng per band). To assess repeatability and
intermediate precision the sample from Grenada region was
used. Repeatability was checked by preparing three concentra-

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Method characteristic

Myristicin

Safrole

Linear range [ng per band]

200800

100700

Correlation coefficient (r)

0.9972

0.9989

Limit of detection [ng per band]

55

20

Limit of quantification [ng per band]

160

70

Instrument precision (RSD [%], n = 10)

0.53

0.35

Repeatability (mean RSD [%], n = 3)

0.37

0.42

Intermediate precision
(mean RSD [%], n = 3)

0.40

0.44

Specificity

Specific

Specific

tions of sample solutions (80, 105, and 120 mg mL1) from this
sample and extracting with methanol as described above. Each
extract was applied in triplicate to the same plate, on the same
day, and analyzed by the above procedure. The intermediate precision of the method was determined in the same way as repeatability, but on three successive days. System suitability was tested by applying myristicin and safrole standard solutions (500
and 400 ng per band, respectively) to the plate six times and
RSD [%] for peak areas and retardation factors (RF) were measured. The accuracy of the method was tested by performing
recovery experiments, by the standard addition method.
Known amounts of myristicin and safrole standard solutions
(1 mg mL1 each), were added at three different levels to
M. fragrans seed powder from Konkan (India), Sri Lanka and
Grenada and the powder was analyzed as described above.
2.5 Analysis of Myristicin and Safrole in Dried Seed Powder
of M. fragrans from Different Locations

Seven replicates of M. fragrans sample solutions (prepared as


described in Section 2.2; 3 L for myristicin and 20 L for saf-

Journal of Planar Chromatography 22 (2009) 6

Short Communications
Table 2
Results from determination of recovery of myristicin and safrole from seed powder of Myristica fragrans Houtt. from the Grenada region.

Component

Level

Amount of sample
weighed [mg]

Amount
added [mg]

Myristicin

1000.3

0.00

1000.5

0.40

1000.2

0.80

2.77 0.015

1000.7

1.20

3.12 0.03

1000.3

0.00

0.12 0.017

1000.5

0.06

0.178 0.04

1000.2

0.09

0.205 0.020

1000.7

0.12

0.237 0.025

Safrole

a)Mean

Mean amount
found [mg]a)
2.0 0.007

Recovery [%]

99.10

2.38 0.01

98.6

SD (n = 7)

Table 3
Results obtained from assay of Myristica fragrans Houtt. from different locations.

Component

Location

Mean weight
of sample [mg]

Myristicin

Konkan (India)

1000.3

Safrole

a)

Mean amount
found in sample [mg]a)

RSD of
peak area [%]

Amount in
powder [%]

4.0 0.03

0.51

0.4

Sri Lanka

1000.5

3.0 0.015

0.43

0.3

Grenada

1000.2

2.0 0.04

0.69

0.2

Konkan (India)

1000.3

0.082 0.01

0.23

0.0082

Sri Lanka

1000.5

0.090 0.042

0.73

0.0090

Grenada

1000.2

0.12 0.035

0.66

0.012

Mean SD (n = 7)

role) were analyzed as described in Section 2.3. The identity of


myristicin and safrole bands from each sample was confirmed
by comparison of RF values with those obtained from myristicin
and safrole standards. The amounts of myristicin and safrole in
each sample solution were determined from the calibration
plots.

3 Results and Discussion


Myristicin has been determined in Carum carvi and Anethum
garveolens [9] by TLC using tolueneethyl acetateformic acid
9.3:0.7:0.1 (v/v) as mobile phase. A TLC method reported for
identification of myristicin from aril or nutmeg used toluene as
mobile phase [8]. Another TLC method reported for identification of myristicin from seed powder of Myristica fragrans
Houtt. used tolueneethyl acetate 9.8:0.2 (v/v) as mobile
phase [9]. In this research work, toluene was used as mobile
phase and enabled efficient resolution of myristicin and safrole
from the other components of the extract. The RF values
obtained for myristicin and safrole were 0.47 and 0.67, respectively.
The UV spectrum of myristicin contained three absorption maxima at 210, 232, and 280 nm. That of safrole contained two
absorption maxima at 200 and 290 nm. The wavelengths of
maximum absorption for myristicin and safrole were 210 and
Journal of Planar Chromatography 22 (2009) 6

290 nm, respectively, so these wavelengths were used for quantification of the compounds.
The calibration plots for myristicin and safrole are shown in
Figures 1 and 2, respectively; the respective residuals plots are
given in Figures 3 and 4. The residuals were distributed randomly around the zero-line, which indicates the correctness of
both calibration plots [10]. The LOD and LOQ for myristicin
and safrole are given in Table 1. The results obtained from measurement of instrumental precision, repeatability, and intermediate precision are also listed in Table 1. RSD [%] for peak areas
and retardation factors (RF) were <2, indicating the method is
reproducible. Results from determination of recovery of myristicin and safrole are given in Table 2. Recovery of myristicin
and safrole from the sample of M. fragrans from Grenada was
99.1 and 98.6%. Recovery of myristicin from the samples from
Konkan (India) and Sri Lanka was 99.6 and 98.7%, respectively. Recovery of safrole from these samples was 98.6 and 99.2%,
respectively. These results indicate the method is accurate.
To study variation of the myristicin and safrole content in relation to the source of the plant, powdered seeds from M. fragrans
from different locations (Konkan (India), Sri Lanka, and Grenada) was analyzed. The largest amounts of myristicin were found
in Konkan sample. The largest amounts of safrole were found in
sample from Grenada. The results obtained from the analysis are
given in Table 3 and Figure 5 shows a developed HPTLC plate,
indicating the bands obtained from myristicin and safrole stan-

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Short Communications

simple, precise, and accurate and can be used for routine quality-control analysis of these seeds.
Acknowledgments

The authors are grateful to Anchrom Analytical (I) Pvt. Ltd,


Mulund, Mumbai, India, for providing facilities to perform this
work.

References
[1] H. Hallstrom, A. Thuvander, J. Nat. Toxins 5 (1997) 186192.
[2] J.S. Pruthi, Minor Spices and Condiments, Crop Management and
Post Harvest Technology, Indian Council of Agricultural Research,
New Delhi, India, 2001.
[3] M.B. Forrester, Hum. Exp. Toxicol. 24 (2005) 56366.
Figure 5
HPTLC plate showing separation of myristicin, B, and safrole, C, in a
methanolic extracts of the seed powder of Myristica fragrans Houtt.
obtained from Konkan region (India), A, Sri Lanka, D, and Grenada, E.

dards and separation of methanolic extracts of seed powder of


M. fragrans from each location.
This method can be applied to samples from other locations for
simultaneous quality control of myristicin and safrole in crude
seed powder of M. fragrans.

[4] A.W. Archer, J. Chromatogr. 438 (1988) 117121.


[5] A. Sjoholm, A. Lindberg, M. Personne, J. Inter. Med. 243 (1998)
327332.
[6] IARC Monographs on the Evaluation of Carcinogenic Risk of
Chemicals to Man, Vol. 10, International Agency for Research on
Cancer, Lyon, 1976, p. 231.
[7] G. Zhou, B. Moorthy, J. Bi, K.C. Donnelly, K. Runderath, Environ.
Mol. Mutag. 48 (2007) 715721.
[8] Quality Standards of Indian Medicinal Plants, Indian Council of
Medical Research, New Delhi, India, Vol 1, 2003, pp. 154157.
[9] K. Dhalwal, V. Shinde, K.R. Mahadik, Chromatographia 67 (2008)
163167.

4 Conclusion
This HPTLC method for simultaneous quantification of myristicin and safrole in seed powder of Myristica fragrans Houtt. is

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[10] A. Mirzaie, A. Hamshidi, S.W. Husain, J. Planar Chromatogr. 20


(2007) 303306.
Ms received: November 27, 2008
Accepted: June 17, 2009

Journal of Planar Chromatography 22 (2009) 6