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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
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
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Peak area
Residuals
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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.
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Method characteristic
Myristicin
Safrole
200800
100700
0.9972
0.9989
55
20
160
70
0.53
0.35
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
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)
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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simple, precise, and accurate and can be used for routine quality-control analysis of these seeds.
Acknowledgments
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.
4 Conclusion
This HPTLC method for simultaneous quantification of myristicin and safrole in seed powder of Myristica fragrans Houtt. is
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