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High Performance Liquid Chromatographic Analysis of Asiaticoside in

Centella asiatica (L.) Urban

Article  in  Chiang Mai Journal of Science · September 2008

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Chiang Mai J. Sci. 2008; 35(3) 521

Chiang Mai J. Sci. 2008; 35(3) : 521-525

www.science.cmu.ac.th/journal-science/josci.html
Short Communications

High Performance Liquid Chromatographic Analysis

of Asiaticoside in Centella asiatica (L.) Urban
Prateek K. Jain and Ram K. Agrawal*
Department of Pharmaceutical Sciences, Dr. H. Gour University, Sagar (M.P.), India.
*Author for correspondence; e-mail: dragrawal2001@yahoo.co.in

Received: 3 March 2008

Accepted: 18 April 2008.

ABSTRACT
A simple, sensitive high performance liquid chromatographic method was developed
for the determination of asiaticoside in Centella asiatica extract. The asiaticoside yielded 1.7
mg/100 mg of the test extract. Calibration graph of asiaticoside showed good linearity in the
concentration range of (32 - 1027 μg/mL), with a correlation coefficient of 0.999. The average
recovery for asiaticoside is 97.5 %. The proposed method is reproducible and statistically
validated. The system was successfully used to investigate the presence of the asiaticoside in
Centella asiatica plant parts.

Keywords: Centella asiatica, asiaticoside, HPLC method.

1. INTRODUCTION
Centella asiatica (Linn) Urban known as most active compounds which can serve as a
Indian pennywort is a prostate, perennial, marker [5, 6] and is used for its standardization.
faintly aromatic herb found wild throughout A few methods such as gravimetric [7] and
India [1,2]. Leaves are used as tonic, memory column chromatography [8] have been
enhancer and also used in the treatment of suggested for the quantitative estimation of
cataract, eye troubles and in fevers and diarrhea asiaticoside which are not very precise,
among children. The plant is reported to sensitive and require multiple step extraction
contain glycosides like brahmosides, and purification. Literatures reveal that HPLC
indocentelloside, asiaticoside, theankuniside methods are also available [9, 10-12].
and isotheankuniside [3]. Asiaticoside, a
trisaccharide triterpene, has been identified as 2. MATERIALS AND METHODS
the most active compound in the plant The roots of the Centella asiatica were
associated with the healing of wounds and picked up in the month of February-March
duodenal ulcers, whilst the triterpene saponins from the University Campus of Dr. H.S. Gour
are also reported to possess immunomodu- Vishwavidyalaya, Sagar (M.P.), collected crude
latory properties [4]. Asiaticoside is one of the drugs were authenticated from the Botany
522 Chiang Mai J. Sci. 2008; 35(3)

Department, Dr. H.S. Gour Vishwavidyalaya, 2.2.1 Standard Preparation

Sagar (M.P.), India. Reference standard Ten milligrams of asiaticoside reference
asiaticoside (purity 98.5%) of Fluka Chemie standard was weighed accurately in to a 10
GmbH, Product No. 43191 was taken as mL volumetric flask. Dissolved in 5 mL of
standard. methanol and then made up to 10 mL with
methanol.
2.1 Chromatographic System 2.2.2 Sample Preparation
Shimadzu high perfor mance liquid Weighed accurately 100 mg of methanolic
chromatographic system equipped with extract in a 100 mL volumetric flask and
LC10A pump with SPD-M 10Avp Photo dissolved in small quantity of methanol and
diode Array Detector or UV detector in made up the volume with methanol.
combination with class LC 10A software.
2.3 Procedure
2.2 Chromatographic Conditions Instrument was adjusted as per conditions
Mobile Phase prescribed. HPLC analysis of the standard and
The mobile phase components were the samples were carried out using above
filtered through 0.2 μm membrane filter mentioned protocol and the chromatograms
before use. Gradient elution was performed were recorded. The retention time and peak
using 0.3% orthophosphoric acid (Soluton A) area of the standard and sample were noted.
and acetonitrile (Solution B) as detailed in Amount of asiaticoside present in the sample
Table 1. was calculated by comparing the sample peak
Table 1. Gradient elution program. area with that of the standard.
Time Buffer (A) Acetonitrile (B)
(min) (mL) (mL) 2.4 Estimation of Asiaticoside
The amount of asiaticoside present in the
0.01 95.0 5
methanolic extract in 20 μL is 0.33 μg;
5.0 80.0 20 therefore 100 mg of extract contain 1.7 mg
15.0 50.0 50 of asiaticoside.

20.0 20.0 80
2.5 Method Validation
23.0 20.0 80 2.5.1 Calibration Curve
30.0 50.0 50 Standard asiaticoside solutions concen-
trations range (32 - 1027 μg/mL) were analyzed
35.0 80.0 20
for studying the linearity and the area count
40.0 95.0 5 obtained for these solutions.
2.5.2 Limit of Detection and Limit of
Other conditions are shown below:
Quantitation
Column : Phenomenex-Luna 5 μ C-18
In order to estimate the limit of detection
(2), Size: 250 x 4.60 mm
(LOD) and lower limit of quantitation
Detector : SPD-M 10Avp Photo diode
(LLOQ), blank methanol was spotted six
array detector/UV Detector
times following the same method. The signal
Wave length : 210 nm
to noise ratio (S/N) was determined. LOD
Flow rate : 1.8 mL / min.
was considered as 3:1 and LLOQ as 10:1.
Injection volume : 20 μL
Chiang Mai J. Sci. 2008; 35(3) 523

2.5.3 Recovery Studies chromatogram of methanolic extract of

Accuracy and precision of the method Centella asiatica. The quantification yielded 1.7
were studied by performing experiments by mg / 100 mg of the test extract. The
standard addition technique. Three different estimation of asiaticoside in Centella asiatica
levels of standards (0.3 μg, 0.6 μg and 0.9 μg) showed in Table 2. The signal to noise ratios
were added to a previously analyzed sample, 3:1 and 10:1 were considered as LOD and
each level being repeated in tripicate. The LLOQ respectively. The LOD and LLOQ
percentage recovery was calculated from were found to be 50 and 200 ng/spot.
amount of drug found. Calibration graph, a plot of peak area
obtained versus asiaticoside concentration,
3. RESULTS AND DISCUSSION showed good linearity in the concentration
A critical analysis of the literatures range of 32 - 1027 μg/mL, (Y = 2055.5X +
revealed that this herb finds widespread use 27975), with a correlation coefficient of 0.999.
in several traditional systems of medicine. The mean percentage recovery obtained for
The number of analytical reports for the asiaticoside was 97.5% showed in Table 3.
determination of asiaticoside is comparatively
small. Only few methods are described in the 4. CONCLUSIONS
literature. All of them show major disadvantage, Standardization of herbal medicines and
the separation time is extremely long (several plant ingredients specially with reference to
hours) or the compounds are not baseline their active marker is time consuming. We
separated and elute more or less with the have tried to standardize Centella asiatica using
injection peak. HPLC for its active compound, asiaticoside.
The HPLC analysis of Centella asiatica The method provides good resolution and
showed asiaticoside peak at retention time separation of asiaticoside from other
11.65 min which was comparable with that constituents of Centella asiatica. The proposed
of standard asiaticoside (retention time, 11.95 HPLC method is rapid, simple and accurate
min). Figure 1 showed chromatogram of for quantitative monitoring of asiaticoside in
standard asiaticoside and Figure 2 showed Centella asiatica.

mV
500
1DetA Ch1

400

300
11.65

200

100

0 5 10 15 20 25 30 35 40 45
min
Figure 1. HPLC chromatogram of standard asiaticoside (128.3 μg/mL); HPLC condition
see text.
524 Chiang Mai J. Sci. 2008; 35(3)

mV
500
1DetA Ch1

400

300

200
11.95

100

0 5 10 15 20 25 30 35 40 45
min

Figure 2. HPLC chromatogram of methanolic extract of Centella asiatica (1 mg/mL); HPLC

condition see text.

Table 2. Estimation of Asiaticoside in Centella asiatica by HPLC.

Replicate Sample Area μg)

Asiaticoside (μ
1 811580 0.36
2 796664 0.32
3 799865 0.32
Average 802703 7852 0.33 0.02
Mean S.D., n = 3

Table 3. Results of Recovery Analysis.

Sample Amount of Amount of Total Total %
asiaticoside asiaticoside asiaticoside asiaticoside Recovery
present in added to A taken (A+B) found D/C x100
μg)
(μ μg)
(μ μg)
(μ μg)
(μ (Mean)
A B C D
Centella 0.3 0.63 0.61 0.04 96.8 6.3
asiatica 0.33 0.6 0.93 0.92 0.01 98.2 1.1
0.9 1.23 1.20 0.09 97.6 7.3
Mean S.D., n = 3.
Chiang Mai J. Sci. 2008; 35(3) 525

ACKNOWLEDGEMENTS [7] Upadhyay S.C., Khosa, R.L., Sharma

The authors are thankful to Indian D.N., Kumar M., and Chansauria J.P.N.,
Council of Medical Research (ICMR), New Total glycoside content and antistress
Delhi and M.P. Council of Science and activity of Indian and Mauritius Centella
Technology (MPCOST) Bhopal, India for asiatica – A comparison, Indian Drugs,
providing financial assistance. 1991; 28(8): 388-389.
[8] Singh B., and Rastogi R.P., A reinves-
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526 Chiang Mai J. Sci. 2008; 35(3)

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Chiang Mai J. Sci. 2008; 35(3) 527

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