The optimal conditions for extraction, hydrolyzation and quantitative determination of the total amounts of two main triterpene acids, asiatic acid, and madecassic acid in Centella asiatica by HPLC and column chromatography are described. Using this method the asiatic acid and madecassic acid content of three Centella asiatica samples collected in Sontay City, Namdinh province, and in Ho Chi Minh City have been screened. The result showed that the contents of both triterpenic acids are higher in Sontay sample and in each sample the content of madecassic acid is higher than that of asiatic acid. Keywords. Centella asiatica, asiatic acid, madecassic acid, quantification

Loc, Tran Van

Phuong Thao, Tran Thi

Quynh Nhu, Vo Thi

Sung, Tran Van

Vietnam Academy of Science and Technology: Journals Online

 Vietnam Journal of Chemistry, International Edition, 54(4): 416-418, 2016 
DOI: 10.15625/0866-7144.2016-00338 
416 
 
 
 
SCREENING FOR THE MAIN TRITERPENIC ACIDS IN CENTELLA 
ASIATICA SAMPLES FROM NORTH AND SOUTH OF VIETNAM 
Vo Thi Quynh Nhu
1
, Tran Thi Phuong Thao
2
, Tran Van Sung
2
, Tran Van Loc
2*
 
1
The Gio Linh Upper secondary school, Quangtri province, Vietnam 
2
Institute of Chemistry, Vietnam Academy of Science Technology  
 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam  
Received 17 June 2016; Accepted for publication 12 August 2016 
 
Abstract 
The optimal conditions for extraction, hydrolyzation and quantitative determination of the total amounts of two 
main triterpene acids, asiatic acid, and madecassic acid in Centella asiatica by HPLC and column chromatography are 
described. Using this method the asiatic acid and madecassic acid content of three Centella asiatica samples collected 
in Sontay City, Namdinh province, and in Ho Chi Minh City have been screened. The result showed that the contents of 
both triterpenic acids are higher in Sontay sample and in each sample the content of madecassic acid is higher than that 
of asiatic acid. 
Keywords. Centella asiatica, asiatic acid, madecassic acid, quantification. 
 
1. INTRODUCTION  
 
Centella asiatica (L.) Urban (syn. Centella. 
coriacea Nannfd., Hydrocotyle asiatica L.) (fam. 
Apiaceae) is a tropical medicinal plant, distributed in 
Southeast Asian countries, India, China, Sri Lanka, 
Madagascar and South Africa [1, 2]. Its leaves are 
edible. Vietnamese use the whole plant as vegetable, 
for making drinks, against fever [3]. In the folk 
medicine this plant has been used since long time 
ago for wound healing, memory enhancement 
effects, treatment of leprosy, as anti-inflammatory, 
antiulcer, hepatoprotective, antioxidant…[4, 5]. The 
main active principles in Centella asiatica are 
triterpenes, triterpene glycosides, flavonoids, sterols 
and essential oil [6]. 
In the searching the plant Centella asiatica with 
high amount of asiatic acid and madecassic acid for 
chemical transformations, we did the screening of 
Centella asiatica collected in Son Tay, Hanoi City, 
Nam Dinh province, and Ho Chi Minh City. 
 
2. EXPERIMENTAL  
 
2.1. Methods and equipment 
 
Column chromatography used silica gel Merck
0.043-0.63 mm; TLC: silica gel G60 F254 precoated 
on the aluminum sheets. HPLC: Alliance series 
2695, detector PDA 2996, Waters, USA. Mobile 
phase: Kanal A: H2O+0.1 % formic acid. Kanal B: 
Acetonitril 1mL/min. gradient. Detector PDA, λ = 
205 nm. 
Analytical balance: Adam AAA 160 L (d = 
0.0001). Establish of standard curve: 25 mg asiatic 
acid (Merck) was placed in a 25 mL standard flash, 
than fill with MeOH to have a stock solution with a 
concentration of 1 mg/mL. The solutions with a 
concentration of 0.05, 0.1, 0.3, 0.5 and 0.7 mg/mL 
were prepared from the stock solution. The prepared 
solutions will be given to the HPLC with the column 
Sunfire-C18Rp (4.6×150 mm), 5 μm. The results in 
table 1 are the averages of three measurements. 
From the results in table 1 the equation of 
regression (y = ax+b) will be established. The 
estimated correlation coefficient is: R = 0.99989992 
and the linear equation of regression is:    
                Y = 10570916. Xi – 60136  
Xi: Concentration of component to be analyzed 
Y: Peak area. 
Similar procedure will be used for madecassic acid 
to estimate the standard curve. 
 
  
VJC, 54(4) 2016                                                                                                                    Tran Van Loc, et al. 
 417 
Table 1: The HPLC date of asiatic acid for establishment of standard curve 
No. Concentration (mg/mL) Peak area Theoretical peak area Retention time (min.) 
1 0.05 455177 468410 19.7 
2 0.1 969416 996956 19.7 
3 0.3 3150939 3111139 19.7 
4 0.5 5271350 5225322 19.7 
5 0.7 7294451 7339505 19.7 
 
 
Fig. 1: The established standard curve for asiatic 
acid 
 
2.2. Plant material 
 
Centella asiatica samples were collected in 
Sontay, Hanoi City, and Namdinh province in April 
2010, the sample from Ho Chi Minh City was 
collected in May 2010 and identified by Mr. Nguyen 
The Anh, Institute of Chemistry, VAST, 18 Hoang 
Quoc Viet, Hanoi.  
2.3. Study the conditions for extraction, 
hydrolyzation and separation of asiatic acid and 
madecassic acid from Centella asiatica 
The Centella asiatica sample (200 gram each) 
was dried at room temperature and extracted three 
times with EtOH/H2O mixture 80:20 (each time 2 h) 
at 80
ο
C. After filtration and evaporation of the 
solvent, the residue was dissolved in 200 mL 
H2O/MeOH 80:20 and the solution was extracted 
with n-hexane for the elimination of fats and 
essential oil. Then it was hydrolyzed with 20 % 
aqueous NaOH solution at 80 
ο
C for 2 hours, let to 
room temperature, extracted with EtOAc. The 
aqueous layer was acidified with HCl to pH = 3-4 to 
give a residue. The residue was filtered, washed with 
H2O and dried. This residue was given to the column 
chromatography and to the HPLC determination of 
the content of asiatic and madecassic acid.  
2.4. Identification of asiatic and madecassic acid 
by TLC 
5 mg of the residue were dissolved in 10 mL 
MeOH and evaluated on the TLC plates (elution 
solvent: CH2Cl2/MeOH 9:1). The developed TLC 
plates were sprayed with vanillin/H2SO4 reagent and 
heated (≈ 110 οC) until the spots were visualized. 
2.5. Quantification by the HPLC method 
 Used the obtained standard curves for asiatic 
acid and madecassic acid. 
2.6. Quantification by column chromatography 
(CC) 
The residue obtained after hydrolyzation with 
NaOH was given to a column chromatography on 
SiO2, eluted with a solvent mixture of 
CH2Cl2/MeOH 95:5 → 90:10 for separation of 
asiatic (1) and madecassic acid (2). 
The optimal conditions for the extraction and 
hydrolyzation to obtain high amount of asiatic acid 
and madecassic acid was investigated. According to 
our study the best conditions for the extraction are 
the followings:  
- Solvent for the extraction from Centella 
asiatica: EtOH/H2O 80:20 (v/v) 
- Temperature for the extraction: 80 
ο
C 
- Time of the extraction: 2 hours. 
For the basic hydrolyzation: 
- Concentration of the aqueous NaOH solution: 
20% (w/w) 
- Time: 2 hours 
- Temperature: 80 
ο
C, reflux 
- Neutralization after hydrolyzation: with 5 % 
HCl solution. 
    
 
 VJC, 54(4) 2016                                                                                Screening for the main triterpenic acids… 
 418 
3. RESULTS AND DISCUSSION 
 
With the procedures described in the 
Experimental part the samples collected in Son Tay, 
Hanoi (RMST), Nam Dinh province (RMND) and 
Ho Chi Minh City (RMHCM) have been analyzed. 
The results of the CC method are given in table 2. 
It was shown from the table 2, that in all three 
Centella asiatica samples the total content of 
madecassic acid is higher than that of the asiatic 
acid. The total content of asiatic acid and madecassic 
acid of Centella asiatica collected in Son Tay, Hanoi 
(RMST) is the highest, and in Ho Chi Minh 
(RMHCM) City is the lowest. The contents of asiatic 
acid and madecassic acid in RMHCM are similar. 
The hydrolyzed residues from three Centella 
asiatica samples were analyzed by the HPLC 
method. The total content in percent of asiatic acid 
and madecassic acid in the residue obtained after the 
hydrolyzation as well as calculated for the dry 
material (Centella asiatica) determined by HPLC 
are given in table 3.  
 
Table 2: The total content of asiatic acid and madecassic acid in three Centella asiatica samples 
Sample Weight 
Extract 
residue (g) 
Residue after 
hydrolyzation (g) 
Asiatic acid (g) (% to 
dry plant material) 
Madecassic acid (g) 
(% to dry plant material) 
RMST 200 30.12 4.25 1.02, (0.51) 1.54, (0.77) 
RMND 200 29.25 4.02 0.92, (0.46) 1.58, (0.79) 
RMHCM 200 25.0 1.67 0.43, (0.21) 0.49, (0.25) 
 
Table 3: The total contents in percent of asiatic acid and madecassic acid in three  
Centella asiatica samples determind by HPLC method 
Constituent RMST 4.25 g residue RMND 4.20 g residue RMHCM 1.67 g residue 
% Area % in dry material % Area % in dry material % Area % in dry material 
Asiatic acid 33.81 0.718 30.49 0.615 28.92 0.241 
Madecassic acid 41.93 0.891 43.02 0.865 28.61 0.239 
 
In comparison of the data in table 2 and 3 it can 
be concluded that the contents of asiatic acid and 
madecassic acid obtained by the CC method were 
lower than estimated by the HPLC method, namely 
about 71 % and 86 % of the HPLC method, 
respectively. 
 
Acknowledgements. This work has been supported 
financially by Vietnam National Foundation for 
Science and Technology Development (NAFOSTED) 
project code 104.01-2012.33. 
 
REFERENCES 
 
1. Pham Hoang Ho. Flora of Vietnam, Youth Publishing 
House, Book 2, p. 477 (2000). 
2. Jamil S. S., Nizami Q., Salam M. Centella asiayica 
(Linn.), Urban: a review in Natural Product 
Radiance, 6(2), 158-170 (2007). 
3. Do Tat Loi. Medicinal Plants and Drugs of Vietnam, 
Publishing House for Science and Technology, 791-
793 (1995). 
4. Tripathi G., Mishra S., Upadhyay P., Purohit S., 
Dubey G. P. Ethnopharmacological Importance of 
Centella asiatica with Special Reference to 
Neuroprotective activity, Asian Journal of 
Pharmacology and Toxicology, 3(10), 49-53 (2015). 
5. Orhan I. E. Centella asiatica (L.) Urban: From 
Traditional medicine to Modern Medicine with 
Neuroprotective Potential, Evidence-Based 
Complementary and Alternative Medicine, 1-8 
(2012). 
6. Brinkhaus B., Lindner M., Schuppan D., Hahn E. G. 
Chemical, pharmacological and clinical profile of the 
East Asian medicinal plant Centella asiatica, 
Phytomedicine, 7(5), 427-448 (2000). 
 
Corresponding author: Tran Van Loc 
Institute of Chemistry, Vietnam Academy of Science Technology  
18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 
E-mail: tvloc@ich.vast.vn; Tel.: 0983992498. 
                                     
  
 
 
 
 


Screening for the main triterpenic acids in Centella asiatica samples from North and South of Vietnam

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Screening for the main triterpenic acids in Centella asiatica samples from North and South of Vietnam

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