Research Article

An improved HPTLC – UV method for rapid estimation of

andrographolide in Andrographis paniculata (Burm. f) Nees
Himanshu Misra1, 2, Manish Soni3, Darshana Mehta1, B. K. Mehta1 and D. C. Jain2, 3*

1
School of Studies in Chemistry and Biochemistry, Vikram University, Ujjain – 456 010, Madhya Pradesh, India.
2
Green Technology Department, Ipca Laboratories Limited, Ratlam –457 001, Madhya Pradesh, India.
3 Mandsaur Institute of Pharmacy, Mandsaur- 458 001, Madhya Pradesh, India.
[e-mail: dc_52@rediffmail.com ; himanshumisra1@rediffmail.com (for correspondence)]

ABSTRACT
A simple, rapid and precise (%RSD<2.5) high-performance thin-layer chromatographic method was
developed for quantitative estimation of a hepatoprotective diterpenoid andrographolide.
Separation was performed on 60 F254 HPTLC plates using chloroform : methanol :: 27 : 3 v/v as
mobile phase for elution of extract components. The determination was carried out in ultra-violet
mode using densitometric absorbance-reflection mode at 232 nm. The concentration of
andrographolide in the whole plant powder was 0.178 (±0.003) %, dry weight basis.
-1
Andrographolide response was found to be linear over a broad range 100 – 2500 ng spot . Limit of
-1
detection and quantification were 40ng and 100ng spot . The developed method is capable of
quantifying smaller as well as higher amounts of andrographolide in its plant raw-material. The
HPTLC method was evaluated in terms of precision, accuracy, sensitivity and robustness.
Keywords: Andrographis paniculata, Acanthaceae, whole plant, HPTLC method, ultra-violet
detection.

INTRODUCTION
Andrographis paniculata (Burm. f) Nees of family Acanthaceae
is traditionally known as kalmegh. The plant is widely used in
ayurvedic and homeopathic systems of medicine. The plant is
also known as ‘king of bitters’ due to its bitter taste and weak
odor. The whole plant is used in medicine and is official in the
Indian Pharmacopoeia (Farooqi & Sreeramu, 2001).
In ayurveda, the drug has been described as antipyretic and
hepatopratective. Cold infusion of the drug is mentioned in
sushruta samhita for fever and liver disorders. Plant drug
contains flavones and lactones. Among lactones
andrographolide (Figure 1) is the main constituent and is active
constituent of the plant. Andrographolide has been isolated in
pure form and it has shown various pharmacological activities
(Caceres et al., 1997). In vitro and in vivo studies suggest that
andrographis has antiinfective, antiviral, antidiarrhoeal,
antipyretic and analgesic activities (Farnsworth &
Bunyapraphatsara; Caceres et al., 1997).
Although some methods was previously developed on
colorimetric (Maiti et al., 1959) and spectrophotometric (Shah
et al., 2007) but they suffers with being multisteps and are not
very precise. Later on few HPTLC / TLC methods (Raina et al.,
2007; Saxena et al., 2000 & Srivastava et al., 2004) developed
but they were utilizing unsafe solvents like, benzene and
toluene and also detection methods were either involves post-
chromatographic derivatization with strong acid or ultraviolet
light with narrower detection range for andrographolide in
crude plant extract. Recently, Akowuah et al. (2006)
established a nice comparison of quantitative analysis of
andrographolide and 14-deoxy-11, 12-
didehydroandrographolide (DIAP) by HPLC and HPTLC
together with LPI assay and free radical scavenging activity of
extracts but HPTLC method suffers with narrow linear range.
Here, we developed an HPTLC – UV method for broad range
detection of andrographolide, which will be very useful for
newer plant variety development to quantify very small as well

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as very large quantities of andrographolide present in
Andrographis paniculata at once.
EXPERIMENTAL
Plant material
Plant material was grown in our experimental field at
Mandsaur institute of pharmacy, Mandsaur (M.P.). Whole
plant was taken, air-dried and pulverized to fine powder of
mesh 22. Sowing was performed in the month of July and
sample taken after 30-35 days before application of first dose of
fertilizer.
Chemicals
Chloroform (99.0 to 99.4% purity) and methanol (99.5%
purity) were purchased from s. d. fine-chem. Limited, Mumbai.
Ethanol was of commercial grade and was utilized after
distillation. Standard andrographolide was previously isolated
and characterized (Saxena et al., 2000).
Apparatus
HPTLC analysis was performed on a Camag’s TLC Scanner 3
controlled by winCATS planar chromatography manager
version 1.4.2 (CAMAG, Switzerland). Drying and concentration
stepts were performed using rotatory evaporator.
Ultrasonicator was used for homogenizing of test and standard
samples.
Preparation of standard solution and calibration curve
50 mg of standard andrographolide was dissolved in methanol,
maintained to mark in a 50 mL volumetric flask and sonicated
for 5 minutes. 1-25 mL of this solution pipetted out and
transferred to seven different 50 mL volumetric flasks, made up
to mark with methanol and sonicated for 5 min. for
homogenizing reference standard of andrographolide. A
calibration curve was plotted between increasing amounts of
andrographolide per spot and their peak area response. A
straight line was obtained between 100 - 2500 ng spot-1.
Extraction and test sample preparation
Three samples each of 5 g fine powder of mesh 22 was
extracted with chloroform, methanol and ethanol (180mL each)
through hot soxhlet extraction for 8 hrs. The extract was
reduced in vacuo and volume made up to 50 mL with methanol
in a volumetric flask. Chloroform extract was made up to the
mark with methanol : chloroform (1:1). These test solutions are
used for quantification purpose of test andrographolide.
Thin-layer chromatographic analysis
Thin-layer chromatography was performed on aluminum
backed HPTLC plates (60 F254, E. Merck, Germany, 200 x 100
mm). Test and standard sample (2µL each) spots were applied
via camag’s Linaomat 5 as 6.0 mm wide bands at the height of
10 mm from base; spots were simultaneously dried with N2 gas
supply on to HPTLC plates. Plates were developed in a camag
twin trough chamber of size more than 200 x 200 mm at 95
mm height from base using chloroform – methanol (27 : 3, v/v)
as mobile phase. Plates were air-dried for complete evaporation
of mobile phase and scanned using camag’s TLC scanner 3
equipped with winCATS software in absorption – reflection
detection mode at 232 nm (Deuterium lamp). Chromatogram
of andrographolide separation was obtained after completion of
scanning (Figure 2).
Research Article
Method validation
Linearity
The linearity of the andrographolide calibration plot (Figure 4)
was evaluated on seven point-scale by spotting increasing
amounts of the andrographolide working standard solution,
starting from 100 - 2500 ng spot-1 (100, 500, 900, 1300, 1700,
2100 and 2500 ng spot-1). For this, a stock solution of 100mg
mL-1 was prepared and then pipette out 1, 5, 9, 13, 17, 21 and 25
mL and transferred to seven different volumetric flask of
volume 50mL, respectively. Volume made to the mark with
methanol and homogenized properly under sonication followed
by manual shaking. 5 µL of each solution spayed over HPTLC
plate and chromatographic separation performed. The method
showed good linearity in the given range with a correlation
coefficient of 0.99727 and the linear regression equation was Y
= 4.032 X + 687.3 (sdv = 4.35).
Precision
Precision of the method determined by three replications of
each sample. The precision (%RSD) of the replications was
found to be less than 2, which is indicative of a precise method
(table 1).
Limit of detection and quantitation (LOD and LOQ)
Limit of detection and quantitation was determined by spotting
increasing amounts (20 – 140 ng; n = 2) of standard
andrographolide solution of concentration 10 µg mL-1 (1mg of
andrographolide per 100 mL) until the average responses were
3 and 10 times of noise for LOD and LOQ respectively. LOD
and LOQ were found to be 40 and 100 ng spot-1 respectively.
Specificity
The developed HPTLC – UV method was found to be specific as
no interfering peak found during detection of andrographolide.
Peaks of andrographolide eluted on to HPTLC plate were found
to be pure, which was also evidenced by peak purity data as
shown in figure 3.
Robustness
Robustness of the method was determined by performing small
variations in mobile phase ratio, height of plate development
and TLC tank saturation time. The results indicated
insignificant differences in assay and thus indicative of a robust
method.
RESULTS AND DISCUSSION
Optimization of extraction
Extraction solvent was standardized for better extraction of
andrographolide using three different solvents (chloroform,
methanol and ethanol) under hot soxhlet extractor and
associated screening of andrographolide content using
presently developed method (table 1). Methanol was found to
be good for better recovery of andrographolide, which was also
supported by previous literature [Srivastava et al., 2004].
Optimization of mobile phase
Mobile phase for the HPTLC separation of andrographolide in
plant extract was optimized using different binary mixtures of
few solvents but chloroform : methanol (27 :3, v/v) was
finalized for better extraction and associated resolution with
the neighboring spots.
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 Research Article

Table 1: Screening of solvents for better extraction

Extraction Wt. of sample (g) Extraction time Extraction % Mean content of (±SD) %RSD
(h) Solvent andrographolide
(n = 3)
Soxhlet 5.0 8.0 Chloroform 0.045 0.001 2.22
Soxhlet 5.0 8.0 Methanol 0.178 0.003 1.69
Soxhlet 5.0 8.0 Ethanol 0.166 0.002 1.20

HO O

CH3
CH2

HO
H3C H

HO
Figure 1: Structure of Andrographolide [C20H30O5 ; Molecular weight = 350.45]

Figure 2: HPTLC Chromatogram of Andrographolide separation

Figure 3: Overlapping UV spectrum of standard andrographolide spot (spot start, middle and end) eluted on to HPTLC plate showing λmax. at
232 nm

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Figure 4: Calibration curve of andrographolide
CONCLUSION
The developed HPTLC method is not only rapid but also
reliable for plants containing low andrographolide contents at
early stages. The method can be utilized for the development of
newer plant varieties as well as for the selection of high-
yielding plant varieties of A. paniculata. Validation parameters
were found to be in good agreement with ideal ones.
ACKNOWLEDGEMENTS
Authors are thankful to Ipca Laboratories Limited for valuable
support and facilities during the course of work.
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Research Article
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