Extraction of Asiaticoside from Centella asiatica :

Effects of solvents and extraction methods

Wan-Joo Kim1), Jae-Duck Kim1)*, Bambang Veriansyah1), Jaehoon Kim1)
Seung- Geun Oh2) and Raymond R Tjandrawinata3)
1) Energy and Environment Research Division, Korea Institute of Science and Technology (KIST), Seoul, 136-791, Korea
2) Division of Chemical & Bioengineering, Hanyang University, Seoul, 133-791, Korea
Supercritical Fluid Lab. 3) PT Dexa Medica Graha Elnusa Building 5th Floor Jl. TB Simatupang Kav. 1B Jakarta 12560 INDONESIA

Centella asiatica Analysis

LCMS Chromatogram centella extract #118-151 RT: 2.14-2.76 AV: 34 NL: 2.32E7
T: + c ESI Full ms [ 200.00-1500.00]
871.41
100
• LC-MS
- Column :C18 5 µm х 25 cm х 4.6 mm
CH 3
90 CH 3

CH3
80
CH 3 CH 3
CH3

OH H
- Ionization mode : API/ES negative & positive mode
OH OH
H OH
70
CH 3
O
HO
CH3 C O H OH
H
H
OH
H
H OH OH
- Drying gas flow : 20 mL/min
H

Relative Abundance
H OH O H H
OH
975.75 CH3 CH2 O H
60 HOCH 2 CH 3
HO
H
H
- Drying gas temperature : 250 oC
OH

Constituents CH2OH CH3

HOH2C CH3

50
521.74
Madecassic acid
- Capillary voltage : 4.5 kv
Main Groups Constituents 40 887.29
991.49 Asiaticoside
Essential oil (0.1% of the plant) Terpene acetate 30 Asiatic acid 593.22

956.71
Gernacrene Caryophyllene 20
216.69 390.79 486.74
505.8 634.98 1221.92
Madecasoside
903.44
298.26 8 1322.00
P-Cynol Pinene 533.12
683.91 813.19
1046.91 1121.98
1479.65
10 758.81 1262.86 1369.76
Flavone derivatives Quercetin glycoside
0
Kaempferol 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z
Sesquiterpenes Caryophyllene
Elemene and bicycloelemene
Trans-farnesene

HPLC Chromatogram
Ermacrene D 1190 • HPLC
Triterpenic steroids Stigmastrol / Sitosterol 200

180 C e nte lla Asiatica

- Column :C18 5µ µm х 25cm х 4.6mm
Triterpenic acid Asiatic acid 990

Madecassic acid
160

140
Asiaticoside - Detector : UV 206nm
Betulinic acid 790
120
- Flow rate : 1.4mL/min

mV
100

80
Asiatic acid
Triterpenic acid sugar esters Asiaticoside - Injection volume : 20µ
µL
mV
60
590
Madecassoside 40

20 - Mobile phase : Gradient

Braminoside 0
0 5 10 15 20 25 30 35 40 45 50
390
T ie m (min) Time (min) Water (%) Acetonitrile (%)
190 00 80 20
30 45 55

Pharmaceutical Effects (10)
0 5 10 15 20 25 30 35 40 45 50 35 45 55
Time(min) 45 80 20
 Wound healing effects
 Anti–ulcer effects
 Antimicrobacterial and antiviral effects
 Immunomodulatory effects
Experiment methods
 Effects on varicose veins
Reference : Matsuda H, Morikawa T, Ueda H, Yoshikawa M (2001) Medicinal Foodstuffs.
XXVII. Saponin constituents Gotu Kola(2): Structures of new ursane- and oleanane-type
Conventional liquid solvent extraction
Heat reflux extraction
Soxhlet extraction
triterpene oligoglycosidesm centellasaponin B, C, and D, from Centella asiatica cultivated
in SriKanka. Chem. Pharm. Bull. 49:113698-1371
- Centella asiatica 3g + 100mL solvent (MeOH, EtOH, Water) - Centella asiatica 3g + 100mL solvent - Centella asiatica 3g + 100mL solvent
- Temperature: Room temperature - Temperature : MeOH(64 oC), EtOH(78 oC) ( MeOH, EtOH, Water mixture)

Asiaticoside distribution percentage - Stir velocity : 1200rpm Water (100 oC) - Temperature : Boiling point
of each tissue of Centella asiatica - Time : 24hr - Time :5 hr - Time :5 hr

Leaves : 82.6%

Nodes : 0 %

Petioles : 15.9 %

Roots : 1.5 %
Reference : O.T. Kim, M.Y. Kim, M.H. Hong” Stimulation of asiaticoside accumulation in

SCCO2 extraction  Pure SCCO2 extraction
the whole plant cultures of Centella asiatica (L/) Urban by elicitors, Physiology and
Biochemistry (2004), 23:339-344
T P Flow rate Time Co- solvent Flow rate Time
P

(oC) (bar) (mL /min) (hr) (%) (mL/min) (hr)

10
T P T P
CO2 Dynamic Co-solvent Static
9 8
50 300 45.2 3 - - -

Application 14
13
P

2
1 T

12  SCCO2 + co solvent extraction

Soap 3
T P Flow rate Time Co- solvent Flow rate Time
15
11
6 (oC) (bar) (mL /min) (hr) (%) (mL/min) (hr)
P
12
T CO2 Dynamic Co-solvent Static
7
Cosmetics Cosmetics 16
80 400 61.3 1 EtOH(65): water(35) =10% 6.1 2
Lotion, skin Mask pack
1. Extractor
2. Jacket
9. Activate Carbon Filter 2nd
10. Condensor
50 400 45.2 1 EtOH(65): water(35) =10% 4.52 2
Centella 3. Pre-Heater
4. Pressure Gauge
11. CO2 Pump
12. Circulator
asiatica 5. Thermocouple 13. CO2 Feed Tank
80 oC
6. Back Pressure Regulator 14. Level Cell 50 oC
extract 7. Separator fraction
8. Activate Carbon Filter 1st
15. CO2 Cylinder
16. Co-solvent vessel

Medicine Medicine
ointment tablet

30 min 180 min 30 min 180 min

Objective of this research Results

20 0.7
H e a t o f re flu x
Comparision of techniques for the extraction E x tra c tio n (a)
Asiaticoside (mg) / Centella asiatica (g)

18 0.6 400bar, dynamic time : 3 h, static time : 1h

(T im e : 5 h r) S x o h le t
E x tra c tio n
- Conventional solvent ( methanol, ethanol, water) 16
(T im e : 5 h r) 0.5
Asiaticoside (mg / g)

14
- Heat of reflux extraction (boiling temperature) 0.4
12
- Soxhlet extraction (ethanol & water mixture) C o n v e n tio n a l
E x tra c tio n
0.3
10
- SCCO2(Supercritical carbon dioxide) extraction (T im e : 2 4 h r)
8 0.2

6 0.1
4 50 oC
0.0
80 oC
Conclusions 2

0
5) 0) 5)
20 40 60 80 100 120 140 160 180 200
H H er OH OH
r (7 (5 (2
eO tO at OH OH te r Et a te at er at er at er
M E W Me Et W
a Me W
2 5 ):
W
5 0 ):
W
7 5 ):
W Time (min)
O H( O H( O H(

The best extraction method of asiaticoside is heat of reflux extraction using ethanol. Et Et Et
S o lve n t o f E x tra c tio n

The polarity of asiaticoside can know using Soxhlet extraction experiment.

The hot ethanol or aqueous ethanol can be alternative solvent. (non-toxic solvent)

Co-solvent SCCO2 extraction can be extracted more effective than pure SCCO2 about asiaticoside.