Key Words
Hemidesmus indicus
2-Hydroxy-4-methoxybenzaldehyde
HPTLC
1 Introduction
Hemidesmus indicus R.Br., Indian sarsaparilla (Anantamul), a
member of the family Asclepiadaceae, is a twining shrub commonly found in India. The woody roots of the plant have a
strong fragrance, and sweet taste with cooling effect. Because of
this typical aroma, the roots are used as a flavoring agent in
sherbets (sweet drinks). They are also used to treat a variety of
ailments or diseases, and are a well-known drug in the
Ayurvedic system of medicine [1] and used as a blood purifier
and also for curing fever, leprosy, rheumatism, and liver disorders [2, 3]. Moreover, root extracts of H. indicus have also
proved to be an effective snake venom antidote [4].
An unusual phenolic compound, 2-hydroxy-4-methoxybenzaldehyde (used as a marker compound) is responsible for the
fragrance of the root [5]. The air-dried material contains approximately 0.225% of essential oil, of which approximately 80% is
2-hydroxy-4-methoxybenzaldehyde, which can be isolated as a
crystalline substance [6]. Recently, 2-hydroxy-4-methoxybenzaldehyde, which is also present in several African medicinal
plants, has been identified as a potent tyrosinase inhibitor [7],
and is thus being used as an ingredient in cosmetic [8] and other
medicinal products, primarily to treat hyperpigmentation [9,
10]. The compound is also known to have antimicrobial [11] and
insecticidal [12] properties.
Literature survey revealed one HPLC method for quantification
of 2-hydroxy-4-methoxybenzaldehyde in the root of Hemidesmus indicus [13], another for simultaneous analysis of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in root tissues of the same plant [14], and a GCMS
method for determination of the chemical composition of
volatile oil of H. indicus [15]. Gas chromatographic assay of 2-
2 Experimental
2.1 Chemicals, Reagents, Materials, and Solutions
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Figure 1
Representative densitogram obtained from the marker compound.
Table 1
Regression data.
Wavelength [nm]
277
2001000
(r2)
0.9972
equationa)
22.971
597.46
9.6
29.09
a)y
Table 2
Results from determination of intra-day and inter-day precision.
RSD [%]
Intra-day
Inter-day
400
0.777
0.2996
600
0.913
1.238
800
0.36
1.2828
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Table 3
Results from recovery studies.
Level [%]
Replica 1
80
100
120
Recovery [%]
Replica 2
99.95
101.8
99.04
100.34
99.54
99.94 0.40
98.94
99.31
100.01 1.55
101.51
101.54
100.69 1.42
The method was validated for linearity, accuracy, precision, limits of detection and quantitation, robustness, and specificity in
accordance with ICH guidelines [20]. Linearity was assessed by
construction of the calibration plot (Section 2.4). Repeatability
and intermediate precision were assessed by measurement of
intra-day and inter-day variation. In the intra-day studies, standard and sample solutions were measured in triplicate on the
same day and RSD [%] was calculated. In the inter-day variation
studies, standard and sample solutions were measured in triplicate on three consecutive days and RSD [%] was calculated.
Accuracy was assessed by measurement of recovery by the
method of standard additions; the method was applied to previously analyzed drug sample to which known amounts of marker
had been added (80, 100, and 120% of the amount known to be
present). Three analyses were performed at each level, and the
results obtained were compared with those expected. The limit
of detection (LOD) of an analytical procedure is the lowest
amount of analyte in a sample which can be detected but not
necessarily quantified accurately. This was calculated by use of
the formula LOD = (3.3 standard deviation of the y-intercept)/(slope of the calibration plot). The limit of quantification
(LOQ) is the lowest amount of analyte in a sample which can be
quantified accurately. This was calculated by use of the formula
LOD = (10 standard deviation of the y-intercept)/(slope of the
calibration plot). Robustness was checked by studying the effect
of slight deliberate changes to the experimental conditions, by
using mobile phases of different composition (tolueneethyl
acetateglacial acetic acid 7:1.8:1 and 6.8:2:1 (v/v). Robustness
was checked at three different concentrations 400, 600, and
800 ng per band. The specificity of the method was assessed by
analysis of standard isolated marker and root powder and checking for the presence of interfering bands from plant constituents
at the RF of the marker. Absorption spectra were also acquired
from and compared with the spectrum obtained from the pure
standard.
Figure 2
Overlain UV spectra of 2-hydroxy-4-methoxybenzaldehyde standard
and spectrum acquired in situ from band at RF 0.7 0.03 from a sample chromatogram.
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References
[1] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, Deharadun,
1991.
[2] M. Prabakan, R. Anandan, T. Devaki, Fitoterapia 71 (2000) 5559.
[3] P.N. Gupta, Lepr. India 53 (1981) 364.
[4] A.I. Alam, B. Auddy, A. Gomes, Toxicon 32 (1994) 15511557.
[5] S. Sreekumar, S. Seeni, P. Pushpangadan, Biotechnol. Lett. 20
(1998) 631635.
[6] A.T. Dutta, S. Ghosh, R.N. Chopra, Arch. Pharm. 276 (1938)
330340.
Figure 3
The amounts of the marker found in the dry powder and in the
hexane extract of root powder were 2.993 and 7.578 mg g1,
respectively. The densitogram obtained from the hexane extract
of the root powder is shown in Figure 3.
4 Conclusion
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