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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
Indian pennywort is a prostate, perennial,
faintly aromatic herb found wild throughout
India [1,2]. Leaves are used as tonic, memory
enhancer and also used in the treatment of
cataract, eye troubles and in fevers and diarrhea
among children. The plant is reported to
contain glycosides like brahmosides,
indocentelloside, asiaticoside, theankuniside
and isotheankuniside [3]. Asiaticoside, a
trisaccharide triterpene, has been identified as
the most active compound in the plant
associated with the healing of wounds and
duodenal ulcers, whilst the triterpene saponins
are also reported to possess immunomodu-
latory properties [4]. Asiaticoside is one of the
most active compounds which can serve as a
marker [5, 6] and is used for its standardization.
A few methods such as gravimetric [7] and
column chromatography [8] have been
suggested for the quantitative estimation of
asiaticoside which are not very precise,
sensitive and require multiple step extraction
and purification. Literatures reveal that HPLC
methods are also available [9, 10-12].
2. MATERIALS AND METHODS
The roots of the Centella asiatica were
picked up in the month of February-March
from the University Campus of Dr. H.S. Gour
Vishwavidyalaya, Sagar (M.P.), collected crude
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
asiaticoside (purity 98.5%) of Fluka Chemie
GmbH, Product No. 43191 was taken as
standard.
2.1 Chromatographic System
Shimadzu high perfor mance liquid
chromatographic system equipped with
LC10A pump with SPD-M 10Avp Photo
diode Array Detector or UV detector in
combination with class LC 10A software.
2.2 Chromatographic Conditions
Mobile Phase
The mobile phase components were
filtered through 0.2 μm membrane filter
before use. Gradient elution was performed
using 0.3% orthophosphoric acid (Soluton A)
and acetonitrile (Solution B) as detailed in
Table 1.
Table 1. Gradient elution program.
Time Buffer (A) Acetonitrile (B)
(min) (mL) (mL)
0.01 95.0 5
5.0 80.0 20
15.0 50.0 50
Ten milligrams of asiaticoside reference
standard was weighed accurately in to a 10
mL volumetric flask. Dissolved in 5 mL of
methanol and then made up to 10 mL with
methanol.
2.2.2 Sample Preparation
Weighed accurately 100 mg of methanolic
extract in a 100 mL volumetric flask and
dissolved in small quantity of methanol and
made up the volume with methanol.
2.3 Procedure
Instrument was adjusted as per conditions
prescribed. HPLC analysis of the standard and
the samples were carried out using above
mentioned protocol and the chromatograms
were recorded. The retention time and peak
area of the standard and sample were noted.
Amount of asiaticoside present in the sample
was calculated by comparing the sample peak
area with that of the standard.
2.4 Estimation of Asiaticoside
The amount of asiaticoside present in the
methanolic extract in 20 μL is 0.33 μg;
therefore 100 mg of extract contain 1.7 mg
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)
2.5.3 Recovery Studies
Accuracy and precision of the method
were studied by performing experiments by
standard addition technique. Three different
levels of standards (0.3 μg, 0.6 μg and 0.9 μg)
were added to a previously analyzed sample,
each level being repeated in tripicate. The
percentage recovery was calculated from
amount of drug found.
3. RESULTS AND DISCUSSION
A critical analysis of the literatures
revealed that this herb finds widespread use
in several traditional systems of medicine.
The number of analytical reports for the
determination of asiaticoside is comparatively
small. Only few methods are described in the
literature. All of them show major disadvantage,
the separation time is extremely long (several
hours) or the compounds are not baseline
separated and elute more or less with the
injection peak.
The HPLC analysis of Centella asiatica
showed asiaticoside peak at retention time
11.65 min which was comparable with that
of standard asiaticoside (retention time, 11.95
min). Figure 1 showed chromatogram of
standard asiaticoside and Figure 2 showed
mV
500
400
300
11.65
200
100
0 5 10 15 20
Figure 1. HPLC chromatogram of standard asiaticoside (128.3 μg/mL); HPLC condition
see text.
523
chromatogram of methanolic extract of
Centella asiatica. The quantification yielded 1.7
mg / 100 mg of the test extract. The
estimation of asiaticoside in Centella asiatica
showed in Table 2. The signal to noise ratios
3:1 and 10:1 were considered as LOD and
LLOQ respectively. The LOD and LLOQ
were found to be 50 and 200 ng/spot.
Calibration graph, a plot of peak area
obtained versus asiaticoside concentration,
showed good linearity in the concentration
range of 32 - 1027 μg/mL, (Y = 2055.5X +
27975), with a correlation coefficient of 0.999.
The mean percentage recovery obtained for
asiaticoside was 97.5% showed in Table 3.
4. CONCLUSIONS
Standardization of herbal medicines and
plant ingredients specially with reference to
their active marker is time consuming. We
have tried to standardize Centella asiatica using
HPLC for its active compound, asiaticoside.
The method provides good resolution and
separation of asiaticoside from other
constituents of Centella asiatica. The proposed
HPLC method is rapid, simple and accurate
for quantitative monitoring of asiaticoside in
Centella asiatica.
1DetA Ch1
25 30 35 40 45
min
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)
ACKNOWLEDGEMENTS
The authors are thankful to Indian
Council of Medical Research (ICMR), New
Delhi and M.P. Council of Science and
Technology (MPCOST) Bhopal, India for
providing financial assistance.
REFERENCES
[1] The Indian Pharmaceutical Codex,
Indigenous drugs, New Delhi, Council
of Scientific and Industrial Research,
Vol. 1, 1953.
[2] British Herbal pharmacopoeia, London,
British Herbal Medicine association, part
2, 1979.
[3] Tiwari N.K., Sharma N.C., Tiwari V., and
Singh B.D, Micropropagation of Centella
asiatica (L.), A valuable medicinal herb,
Plant Cell Tissue and Org. Cult., 2000; 63:
179-185.
[4] Plohmann B., Bader G., Streich S., Hilter
K., and Franz G., Immunomodulatory
effects of triterpenoid saponins, Eur. J.
Pharm. Sci., 1994; 2: 12-17.
[5] Tyler V., Brady L., Robbers J.
Pharmacognosy, 8th Edn, Lea and Febiger,
Philadelphia, 1981.
[6] Kartnig T., Clinical applications of Centella
asiatica (L.) Urb., Herbs, Spices, Medicinal
plants, Vol. 3, 1988.
525
[7] Upadhyay S.C., Khosa, R.L., Sharma
D.N., Kumar M., and Chansauria J.P.N.,
Total glycoside content and antistress
activity of Indian and Mauritius Centella
asiatica – A comparison, Indian Drugs,
1991; 28(8): 388-389.
[8] Singh B., and Rastogi R.P., A reinves-
tigation of the triterpenes of Centella
asiatica, Phytochem., 1969; 8: 917-921.
[9] Verma R.K., Bhartariya K.G., Gupta
M.M., and Kumar S., Reverse phase high
performance liquid chromatography of
asiaticoside in Centella asiatica, Phytochem.
Anal., 1999; 10: 191-193.
[10] Chauhan S.K., Singh B., and Agarwal S.,
A HPLC determination of asiaticoside
in Centella asiatica, Indian J. Nat. Prod.,
2003; 19 (4): 21-23.
[11] Schaneberg B.T., Mikell J.R., Bedir E.,
and Khan I.A., An improved HPLC
method for quantitative determination of
six triterpenes in Centella asiatica extracts
and commercial products, Pharmazie,
2003; 123-125.
[12] Inamdar P.K., Yeole R.D., Ghogare A.B.,
and De Souza N.J., Determination of
biologically active constituents in Centella
asiatica, J. Chromatogr. A., 1996; 742:127-
130.
526 Chiang Mai J. Sci. 2008; 35(3)

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

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