INTRODUCTION:
Andrographolide is
a labdane diterpenoid that
is the main bioactive component of the medicinal plant Andrographis paniculata[1,2]
(kalmegh).
Andrographolide is an extremely bitter substance
extracted from the stems and leaves of the andrographis paniculata[3,4] which is grown for medicinal purposes in China, Srilanka and India. Chemically Andrographolide
is 3-[2-[decahydro-6-hydroxy-5- (hydroxymethyl)-5,8a-
dimethyl-2-methylene-1- napthalenyl] ethylidene] dihydro-
4-hydroxy-2(3H)- furanone. It is used as
anti-inflammatory, immunosuppressant and neuroprotective
effect. It is also used in the treatment of moderate to severe pain. So a
suitable and validated method has to be available for analysis of drugs in
bulk, and biological samples. According
to literature survey revealed that HPTLC[5-7],
HPLC[8-12], Micellar Electrokinetic
Chromatography[13] and Spectrophotometry [14]
methods were published and then it becomes essential to develop a simple and
sensitive colorimetric method for the estimation of andrographolide
in kalmegh extract.
Figure: 1. Structure of Andrographolide
Thus the present study was
undertaken to develop a simple colorimetric method of andrographolide
by using picric acid in an alkaline medium, and validated the method as per ICH
guidelines.[15]
MATERIALS AND
METHODS:
UV-Visible spectrophotometer of Thermo
scientific, Model: Genesys 10 Bio with Spectral band
width of 3nm and 1 cm matched quartz cell was used. Pure drug of Andrographolide was supplied by Laila
Impex as gift sample.
Chemicals and
Reagents:
AR grade methanol, 1% Picric acid in
methanol and 10% NaOH
solution.
Optimization of
parameters for colorimetric method:
All the optimization parameters were
estimated at room temperature. Andrographolide was
found to yield a red-orange colored product with picric acid in alkaline medium
and had absorbance maxima (λmax) at
481nm. Therefore, investigations were carried out to establish the most
favorable conditions for the formation of colored product. The influence of the
ratio of picric acid and NaOH as well as the volume
of reagent mixture on the reaction has been studied. Different ratios and different volumes were
tried for optimization, by varying one parameter at a time. The optimum ratio
of Picric acid: NaOH is 80:20, similarly optimum
volume of reagent mixture was found to be 5 ml respectively. The optimum ratio
and volume were selected on the basis of their ability to give maximum
absorbance and also the color
stability of newly
formed complex was measured.
Procedure:
Preparation of the standard stock solution:
A stock solution (100 µg/mL) of andrographolide was
prepared by dissolving 5 mg of drug in 50 mL of
methanol. From the above solution 3mL was transferred into a 10mL volumetric
flask, to this flask 5mL of picric acid and NaOH
reagent mixture was added. Finally the volume was made up to the mark with
methanol. The flask was kept aside for 20min. The absorbance of the colored chromogen was measured at 481 nm against reagent blank
after 20 min.
Preparation of sample solution:
5mg of kalmegh
sample was weighed accurately and transferred to a 50 mL
volumetric flask, add 30mL of methanol and sonicated
for 10 min for dissolving of andrographolide and then
the sample solution was filtered and diluted to 50 ml with methanol.
METHOD VALIDATION:
Validation is a process of establishing
documented evidence, which provides a high degree assurance that a specific
activity will consistently produce a desired result, or a product meeting its
predetermined specifications and quality characteristics. The method was
validated for different parameters like Linearity, Accuracy, Precision, Limit
of detection (LOD) and Limit of quantification (LOQ).
Linearity:
Various aliquots were prepared from the
stock solution (100µg/mL) ranging from 10-100 µg/mL. The samples were analyzed with the help of a UV-VIS
Spectrophotometer against its blank. The correlation coefficient was found to
be 0.9991.
Accuracy:
To check the accuracy of the developed
method recovery studies were carried out by preparing solutions of different
concentrations, i.e., 80, 100, 120% in which the sample concentration was kept
constant and the amount of pure drug was varied respectively. The solutions
were prepared in triplicate and the accuracy was indicated by % Recovery.
Precision:
The precision of the method was
demonstrated by intra-day and inter-day variation studies. In the inter-day
variation study, the solutions of same concentration were prepared and analyzed
for three consecutive days, and the absorbances were
recorded. In the intra-day variation study, the solutions of the same
concentration were prepared and analyzed thrice a day (morning, afternoon, and
evening). The result was indicated by % RSD.
LOD and LOQ:
The LOD is the detection limit of an
individual analytical procedure is the lowest amount of analyte
in a sample, which can be detected, but not necessarily quantitated
as an exact value. The LOQ is the concentration that can be quantitated
reliably with a specified level of accuracy and precision. The LOD & LOQ
were calculated using the formula involving the standard deviation of response
and the slope of the calibration curve.
Where
σ - Standard deviation of the
response,
s – Slope ratio curve
RESULTS & DISCUSSION:
The proposed colorimetric method was
simple, rapid and precise for estimation of androgrpholide
in kalmegh extract. In this method initially picric acid reacts with the sodium hydroxide
which forms sodium picrate then the
formed sodium picrate was condensed with the
γ-lactone ring of andrographolide
results in the formation of reddish orange colour
complex which could be measured at 481nm.The absorption spectrum of andrographolide was shown in Fig.2.
Fig: 2. Absorption
Spectrum of Andrographolide by Colorimetry
The optical characteristics and the data
concerning the proposed method were represented in Table 1. The % assay was found to be 48.69 in kalmegh
extract and the results were incorporated in Table: 2. It obeyed beer’s law in the concentration range of
10-100µg/mL with a correlation coefficient of 0.9999
(Fig. 3). The precision of the method expressed as %
RSD of intraday and interday were given in Table:
3 & 4. The recovery studies were satisfactory and the percentage of
drug recovered was in the range of 99.72-101.48% included in Table: 5. The sensitivity was estimated
in terms of limit of quantification (LOQ), the smallest amounts detected were
estimated in terms of limit of detection (LOD) the results also shown in Table:6. The recovery studies were
carried out for the developed method by the addition of standard drug solution
of andrographolide to pre-analyzed sample and the %
recovery was found to be in the range of 99.98 to 100.01%. Thus the proposed
method was found to be simple, sensitive, accurate, and precise hence it was
successfully applied for determination of andrographolide
in the extracts, newly formulated ayurvedic
formulations.
Table: 1.
Optimum Conditions of the Proposed Method
|
Parameters |
Optimum range |
Conditions in procedure |
|
Ratio
of reagent |
95:05
to75:25 |
80:20 |
|
Volume
of reagent |
1-7ml |
5ml |
|
Stability
of Colour |
10-60min |
20min |
|
Temperature |
28
± 2°C |
28
± 2°C |
Table: 2. Assay result
|
Sample name |
Quantity of Sample taken (mg) |
Amount of Andrographolide
found* (mg) |
% Assay* |
|
Kalmegh Extract |
5 |
2.48 |
48.96 |
*Average of six determinations
Table:
3. Intraday precision
|
Concentration (µg/mL) |
%RSD* |
Average
%RSD* |
|
30 |
Abs1
Abs2 Abs3 0.7 0.83 0.64 |
0.723 |
*Average
of six determinations
Table: 4. Inter-day precision
|
Concentration(µg/mL) |
%RSD* |
Average
%RSD* |
|
30 |
Day1
Day2 Day3 0.8
1.10 0.75 |
0.88 |
*
Average of six determinations
Table: 5. Recovery results
|
Sample
name |
Concn of Sample (µg/mL) |
Level of addition (%) |
Amount
of drug added* (µg/mL) |
%
Recovery |
|
Kalmegh |
30 |
80 |
24 |
101.48 |
|
30 |
100 |
30 |
100.13 |
|
|
30 |
120 |
36 |
99.72 |
Table:
6. Validation Parameters
|
Parameters |
Result |
|
Absorption
maxima (λmax) |
481nm |
|
Beer’s
law limits |
10-100µg/mL |
|
Correlation
coefficient(R |
0.9998 |
|
Intraday
Precision(%RSD) |
0.723 |
|
Interday
Precision(%RSD) |
0.88 |
|
Slope |
0.01176 |
|
LOD |
1.0
µg/mL |
|
LOQ |
3.0µg/mL |
Figure: 3. Calibration curve of Andrographolide
by Colorimetry
CONCLUSION:
Unlike GC and
HPLC techniques, Spectrophotometry is simple and
inexpensive. The Spectrophotometric methods require simple reagents, which an
ordinary analytical laboratory can afford and the Procedures do not involve any
critical reactions. Moreover the proposed method is found to be simple,
sensitive, accurate, and with good precision, thus, this approach could be
considered for the analysis of this drug in the quality control laboratories.
REFERENCES:
1. Chaturvedi GN. Clinical
studies on kalmegh (Andrographis
paniculata) in infectious hepatitis. Journal of the
International Institute of Ayurveda, 1983,
2(4):208–211.
2. Chao
W-W., Lin B.-F. Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian) Chinese
Medicine 2010.5:17, 1-15.
3. Chandana Majee.
HPLC Method Development and Characterization of Bio-Active Molecule Isolated
from Andrographis paniculata.
International Journal of PharmTech Research,
2011; 3(3), 1586-1592.
4. Dimpy Das Borooah.
Column Chromatography for Isolation of Andrographolide from In Vitro Grown Medicinal Plant-Andrographis paniculata
Nees, International Journal of Applied Biology
and Pharmaceutical Technology . 2011; 2 (3),571-575.
5. Monika Jadhao.
Estimation of Andrographolide in Herbal Powder and Polyherbal Asava by HPTLC,
International Journal of Pharma and Bio Sciences.
2010; 1(4):242-245.
6. S. H. Mishra. Simultaneous Estimation of Andrographolide
and Wedelolactone in Herbal Formulations, Indian
Journal of pharmaceutical sciences. 2008;70(5):689-693.
7. K. Vijaykumar.
Estimation of Adrographolide
in Andrographis paniculata
Herb, Extracts and Dosage forms, International Journal of Applied
Science and Engineering 2007; 5(1), 27-39.
8. TaoXu. Simultaneous Determination of
Four Andrographolides in Andrographis paniculata Nees by Silver Ion RP-HPLC, Journal of
Chromatographic Science. 2008;
46(8):747-50.
9. K. Senthil
Kumaran. An HPLC Method for the estimation of Andrographolide in Rabbit serum , Indian Journal of
Pharmacology. 2003; 35: 109-112.
10. Meenu Sharma, Aakanksha
Sharma And Sandeep Tyagi.
Quantitative HPLC analysis of andrographolide in Andrographis paniculata at two
different stages of Life cycle of plant.
Acta Chimica& Pharmaceutica. Indica. 2012;
2(1): 1-7.
11. A. Srivastava, H. Misra, R. K. Verma and M. M.
Gupta. Chemical Finger Printing of Andrographis Paniculata using HPLC, HPTLC and Densitometry. Phytochem Anal. 2004 ;15(5):280-285.
12. L. Chen, H. Jin, L. On-line
Coupling of Dynamic Microwave-Assisted Extraction with High Performance Liquid Chromatography
for Determination of Andrographolide and Dehydro Andrographolide in Andrographis paniculata Nees, J. Chrmatogr.
A.2007;1140:71-77.
13. Q. Du, G. Jerz
and P. Wiinterhalter, Separation of Andrographolide and Neoandrographolide
from the Leaves of Andrographis Paniculata
by Micellar Electrokinetic
Chromatography, J.Chromatogr. A.2003;984:147-151.
14. Chantana Aromdee, Wichitchote,
P. and Jantakun, N. Spectrophotometric determination of total lactones in Andrographis paniculata Nees, Songklanakarin J.
Sci. Technol. 2005; 27(6) :
1227-1231.
15. ICH Guidelines Q2B, Validation
of Analytical Procedure: Definitions, published in March 1996, Geneva, Switzerland.
Received on 20.06.2014 Accepted on 28.06.2014
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