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Andrographolide determination of Andrographis paniculata extracts, ethyl

acetate fractions and tablets by thin layer chromatography

Article · December 2015

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Journal of Chemical and Pharmaceutical Research, 2015, 7(12): 557-561

ISSN : 0975-7384
Research Article CODEN(USA) : JCPRC5

Andrographolide determination of Andrographis paniculata extracts, ethyl

acetate fractions and tablets by thin layer chromatography
Achmad Fuad Hafid1,3, Bachtiar Rifai5, Lidya Tumewu3, Endang Widiastuti2, Lilis
Dachliyati2, Riesta Primaharinastiti4 and Aty Widyawaruyanti1,3
1
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya,
Indonesia.
2
PT. Kimia Farma Tbk, Bandung, Indonesia
3
Center for Natural Product Medicine Research and Development, Institute of Tropical Disease, Universitas
Airlangga, Surabaya, Indonesia
4
Department of Analytical Chemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
5
Postgraduate student, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia
_____________________________________________________________________________________________

ABSTRACT

The purpose of this study was to develop a simple, sensitive and validated analytical method to determine
andrographolide in Andrographis paniculata. Simple Thin Layer Chromatograpy (TLC) method was performed to
determine andrographolide in extracts, ethyl acetate fractions and tablets of A. paniculata with silica gel 60 F254 as
stationary phase. Linear ascending development was carried out with chloroform: methanol (9:1 v/v). The Rf value
of andrographolide was found to be 0.39. Camag TLC Scanner 3 was used for peak analysis and scanned at
wavelenght 228 nm. The correlation coefficient of the calibration curve was 0.998 in the range of 0.08 µg to 0.60
µg. The recovery of the method was 106.74% - 109.72%. The relative standard deviation was 1.52 - 4.48%.
Application this method for andrographolide determination in sample of phytopharmaceutical product development
showed andrographolide content in 96% ethanol extract, 70% ethanol extract, ethyl acetate fraction-96, ethyl
acetate fraction-70, ethyl acetate fraction-96 tablet and ethyl acetate fraction-70 tablet were 13.94%, 10.12%;
27.22%, 33.12%, 6.51% and 6.10%, respectively. The proposed analytical method was simple, selective and valid
for determination of andrographolide in extracts, ethyl acetate fractions and tablets of A. paniculata.

Keywords: TLC, andrographolide, A. paniculata, extract, ethyl acetate fraction, tablet.

_____________________________________________________________________________________________

INTRODUCTION

Andrographis paniculata (family Acanthaceae) was locally known as sambiloto in Indonesia. This plant was widely
grown in tropical area especially South Asia and Southeast Asia covering India, Srilanka, Pakistan and Indonesia
[1]. A. paniculata was one of the most popular medicinal plants which used traditionally to treats infectious disease
and fever. Some pharmacological activity of A. paniculata already reported which were antimicrobe, antiprotozoa,
antiinflammation, antioxidant, immunostimulant, cytotoxic, antidiabetic [2] and antimalarial [3, 4, 5, 6, 7]. The
major component of A. paniculata was andrographolide. Andrographolide was a colorless crystal form of diterpene
lactone compound, one of compounds which take a role in antimalarial activity of A. paniculata [6,8]. Our previous
study founded that ethyl acetate fraction containing andrographolide as the marker compound in
phytopharmaceutical products at a dose of 20.1 mg/kg mice body weight per day was shown inhibition of
Plasmodium berghei growth with inhibition range of 70.15 to 80.35% [6]. Marker compound was needed in the
standardization process of herbal extract or natural product which was very essential to ensure the consistency of

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Achmad Fuad Hafid et al J. Chem. Pharm. Res., 2015, 7(12):557-561
______________________________________________________________________________
quality and biological activity. Therefore, determination of andrographolide in A. paniculata was extremely needed.
Especially in our research to ensure quality of phytopharmaceutical product development with raw material ethyl
acetate fraction was used for tablet formulation as antimalarial.

Some analysis method of andrographolide in A. paniculata were reported including High Performance Liquid
Chromatography (HPLC) [9], HPLC- Evaporative Light Scattering Detector [10], High Performance Thin Layer
Chromatography (HPTLC) [8,11], HPLC and HPTLC [12]. However, a simple, sensitive and inexpensive method
for routine analysis of andrographolide especially in extract, ethyl acetate fraction and product of A. paniculata still
preferred. In this study, we develop a simple, sensitive and valid TLC method to determine andrographolide in
ethanol extract, ethyl acetate fraction and product of A. paniculata.

EXPERIMENTAL SECTION

Material
Andrographis paniculata Nees herb powder was obtained from PT. Kimia Farma Tbk., Andrographolide standard
was purchased from Sigma-Aldrich, chloroform (Merck), methanol (Merck), ethanol (Merck), pure water was
obtained by the Elix Millipore system.

Extraction method
1,000 g of A. paniculata dried powder was extracted using 6 l of 96% ethanol as a solvent (solvent ratio 1:6).
Maceration was conducted for 60 minutes with temperature 50 oC. Extract then filtered and residue was extracted
again using the same treatment. Total solvent which used for extraction was 12 l (2x6 l). Extract was evaporated
using rotary evaporator until ±4.8 l concentrated extract was obtained. Concentrated extract will further fractionate
using ethyl acetate. Meanwhile, dried 96% ethanol extract was obtained by concentrate the liquid extract using
rotary evaporator and keep the extract in oven with temperature 40oC for 48 hours. The similiar procedure was
conducted using 70% ethanol as a solvent to obtain 70% ethanol extract.

Fractionation method
Concentrated extract was further fractionated using ethyl acetate with composition of extract:water:ethyl acetate was
1:2:1 v/v/v.

Sample preparation for TLC

Sample 70% ethanol extract, 96% ethanol extract, ethyl acetate fraction-70 and ethyl acetate fraction-96 were
weighted 15 mg each and diluted in 10 ml of 96% ethanol. Liquid samples were transfered to 25 ml volumetric
flask, sonicated for 10 minutes and added 96% ethanol until 25 ml. Samples were spotted 2 µl on TLC plate.

Sample ethyl acetate fraction-70 tablet was weighted 90 mg and ethyl acetate fraction-96 tablet was weighted 60 mg
and each diluted in 10 ml of 96% ethanol. Liquid samples were transfered to 25 ml volumetric flask, sonicated for
10 minutes and added 96% ethanol until 25 ml. Samples were spotted 2 µl on TLC plate.

Chromatography conditions
Separation of andrographolide was performed on silica gel 60 F254 plate (20x10 cm), sample and standard reference
was applied 2 µl and distance between spot 10 mm. Eluation was performed using CAMAG Automated Developed
Chamber (ADC). The Composition of mobile phase was chloroform:methanol (9:1 v/v). The optimized chamber
saturation was 20 minutes at room temperature, plate preconditioning time 3 minutes, migration distance 80 mm,
drying time in the ADC system was 5 minutes. The plate was scanned over wavelenght of 200-400 nm with
CAMAG TLC Scanner 3. The maximum absorbance was found to be 228 nm.

Curve Calibration of Andrographolide

Stock solution of andrographolide standard (1000 ppm) were prepared in ethanol. A series of standard curves
concentration of 40 ppm, 50 ppm, 100 ppm, 150 ppm, 200 ppm, 300 ppm were prepared. The stock solution was
spotted 2 µl to give andrographolide content of 0.08 µg, 0.1 µg, 0.2 µg, 0.3 µg, 0.4 µg and 0.6 µg. The data of spot
area versus standard concentration was treated by linear least square regression analysis.

Selectivity
The selectivity of the method was ascertained by analyzing andrographolide standard and samples. The spot for both
andrographolide standard and andrographolide in sample was confirmed by comparing the Rf values and similiarity
index of the UV Spectra.

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Achmad Fuad Hafid et al J. Chem. Pharm. Res., 2015, 7(12):557-561
______________________________________________________________________________
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
In order to determine detection and quantification limit, standard concentrations in the lower part of the linear range
of the calibration curves were used. Stock solution of 500 µg/ml was prepared in ethanol. A series of standard curve
concentrations of 25 ppm, 40 ppm, 50 ppm, 70 ppm, were prepared from stock solution. The corresponding slope (b)
and residual standard deviation (Sy) values were used to calculate LOD and LOQ with the following equation.

;
Accuracy
Accuracy of the proposed method was determined by standard addition technique. Three different concentrations
(80%, 100% and 120%) of standard were added to a previously analyzed sample and the analysis was performed in
triplicates.

Precision intraday
Precision of an analytical method is expressed by % Relative Standard Deviation (% RSD) of series of
measurement. Three different concentrations (80%, 100% and 120%) of standard were added to a previously
analyzed sample and the analysis was performed in triplicates.

RESULTS AND DISCUSSION

Selectivity
Selectivity test showed that Retention factor (Rf) of andrographolide standard was 0.38 and andrographolide in
sample ethyl acetate fraction of A. paniculata showed the similiar Rf of 0.38. Overlay ultraviolet (UV) spectra of
andrographolide in sample ethyl acetate fraction and andrographolide standard showed good correlation with
similiriaty index of 0.9874 (Fig.1). Overlay UV spectra of andrographolide in sample ethyl acetate fraction addition
with andrographolide standard and andrographolide standard also showed good correlation with similarity index of
0.9956 (Fig.2).

(A) (B)
Figure 1. The UV Spectra of andrographolide standard (A), overlay UV spectra of andrographolide in sample ethyl acetate fraction (2)
and andrographolide standard (1) (B) using TLC Scanner at wavelength 228 nm

Linearity
The correlation of andrographolide standard concentration versus response area detector showed linier correlation at
concentration range of 40-300 ppm with regression equation Y = 9882 X + 443.9 and R2 = 0.9968 (r = 0.9984).

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were calculated using corresponding slope and residual standard deviation equation. The results
showed that LOD was 0.010 µg and LOQ was 0.035 µg.

Accuracy and Precision

Accuracy was conducted by sample addition method. Sample ethyl acetate fraction was added with andrographolide
standard using three different concentrations which were 80%, 100% and 120%. Accuracy analysis was done in
triplicates and % recovery was calculated. Accuracy analysis results was showed in table 1.

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Achmad Fuad Hafid et al J. Chem. Pharm. Res., 2015, 7(12):557-561
______________________________________________________________________________
Precision also conducted by sample addition method. Sample ethyl acetate fraction was added with andrographolide
standard using three different concentrations which were 80%, 100% and 120%. Precision analysis was done in
triplicates and % RSD was calculated. Precision analysis results was showed in table 1.

2
1

(A) (B)
Figure 2. Spectra of andrographolide standard (A), overlay UV spectra of andrographolide in sample ethyl acetate fraction addition with
andrographolide standard (2) and andrographolide standard (1) (B) using TLC Scanner at wavelength 228 nm

Table 1. Accuracy and precision analysis results

Addition concentration % Recovery Average of % Recovery % RSD

101.35
80% 110.51 106.74 4.48
108.36
108.47
100% 111.61 109.72 1.52
109.08
107.49
120% 104.73 106.87 1.78
108.38

Determination of andrographolide in sample

Sample which used in this study for andrographolide analysis were 96% ethanol extract, 70% ethanol extract, ethyl
acetate fraction-96, ethyl acetate fraction-70, ethyl acetate fraction-96 tablet and ethyl acetate fraction-70 tablet. The
96% ethanol extract was obtained from extraction of A. paniculata dried powder using 96% ethanol as a solvent. It
was further fractionated using ethyl acetate to obtain ethyl acetate fraction-96. The formulation of ethyl acetate
fraction-96 was conducted to produce ethyl acetate fraction-96 tablet. Meanwhile, 70% ethanol extract was obtained
from extraction of A. paniculata dried powder using 70% ethanol as a solvent. The 70% ethanol extract was further
fractionated using ethyl acetate to obtain ethyl acetate fraction-70. The formulation of ethyl acetate fraction-70 was
conducted to produce ethyl acetate fraction-70 tablet.

Table 2. Results of andrographolide content in samples

Sample Dry weight of Sample (g) Content of Andrographolide (%)

96% ethanol extract 0.2048 13.94
70% ethanol extract 0.2405 10.12
Ethyl acetate fraction-96 2.0379 25.29
Ethyl acetate fraction-70 2.5375 30.11
Ethyl acetate fraction-96 tablet 0.6506 6.51
Ethyl acetate fraction-70 tablet 0.6531 6.10

CONCLUSION

Simple, selective and validated TLC method can be applied for determination of andrographolide content in ethanol
extracts, ethyl acetate fractions and ethyl acetate fraction tablet of A. paniculata. This method can be adopted as
routine analysis method in pharmaceutical industry for determination of andrographolide content in herbal product
material extract or fraction.

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 Achmad Fuad Hafid et al J. Chem. Pharm. Res., 2015, 7(12):557-561
______________________________________________________________________________
Acknowledgement
This research was funding by DIPA DITLITABMAS, Directorate General of Higher Education Research and
Technology, Republic of Indonesia, contract No. 266/UN3.14/LT/2015.

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