Extraction of Extraction of AsiaticosideAsiaticoside from from CentellaCentella asiaticaasiatica : :
Effects of solvents and extraction methods Effects of solvents and extraction methods WanWan--JooJoo KimKim1)1), , JaeJae--Duck KimDuck Kim1)*1)*, , BambangBambang VeriansyahVeriansyah1)1), , JaehoonJaehoon KimKim1)1)
SeungSeung-- GeunGeun OhOh2)2) and Raymond R Tjandrawinataand Raymond R Tjandrawinata3)3)
1) Energy and Environment Research Division, Korea Institute of 1) Energy and Environment Research Division, Korea Institute of Science and Technology (KIST), Seoul, 136Science and Technology (KIST), Seoul, 136--791, Korea791, Korea
2) Division of Chemical & Bioengineering, 2) Division of Chemical & Bioengineering, HanyangHanyang University, Seoul, 133University, Seoul, 133--791, Korea791, Korea
3) PT 3) PT DexaDexa MedicaMedica GrahaGraha ElnusaElnusa Building 5th Floor Building 5th Floor JlJl. TB . TB SimatupangSimatupang KavKav. 1B Jakarta 12560 INDONESIA. 1B Jakarta 12560 INDONESIA
Centella asiatica
� ConstituentsConstituentsMain Groups
Terpene acetate
Gernacrene Caryophyllene
P-Cynol Pinene
Quercetin glycoside
Kaempferol
Caryophyllene
Elemene and bicycloelemene
Trans-farnesene
Ermacrene D
Stigmastrol / Sitosterol
Asiatic acid
Madecassic acid
Betulinic acid
Asiaticoside
Madecassoside
Braminoside
Essential oil (0.1% of the plant)
Flavone derivatives
Sesquiterpenes
Triterpenic steroids
Triterpenic acid
Triterpenic acid sugar esters
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
triterpene oligoglycosidesm centellasaponin B, C, and D, from Centella asiatica cultivated
in SriKanka. Chem. Pharm. Bull. 49:113698-1371
�Wound healing effects
� Anti–ulcer effects
� Antimicrobacterial and antiviral effects
� Immunomodulatory effects
� Effects on varicose veins
� Pharmaceutical Effects
� Application
Centella
asiatica
extract
Cosmetics
Lotion, skin
Soap
Medicine
ointment
Cosmetics
Mask pack
Medicine
tablet
Objective of this research
Comparision of techniques for the extraction
- Conventional solvent ( methanol, ethanol, water)
- Heat of reflux extraction (boiling temperature)
- Soxhlet extraction (ethanol & water mixture)
- SCCO2(Supercritical carbon dioxide) extraction
centella extract #118-151 RT: 2.14-2.76 AV: 34 NL: 2.32E7T: + c ESI Full ms [ 200.00-1500.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
871.41
975.75
521.74
887.29991.49
593.22
956.71486.74216.69 390.79
1221.92634.98 903.44298.26 1322.00813.19683.91 1046.91 1121.98533.12 1479.651369.76758.81 1262.86
Asiaticoside
Madecasoside
Madecassic acid
505.8
8
Asiatic acid
CH3
OH
OH
HOCH2CH3
CH3 CH3
CH3
OH
O
CH3
HO
HO
HOH2C
C O
H
OH H
H OH
OH
HH
CH2 O
H
OH H
H OH
HH
CH2OH
O
OH OH
H H
H
HOH
CH3
CH3
CH3
CH3
CH3
CH3
(10)
190
390
590
790
990
1190
0 5 10 15 20 25 30 35 40 45 50
Time(min)
mV
Cente lla Asiatica
0
20
40
60
80
100
120
140
160
180
200
0 5 10 15 20 25 30 35 40 45 50
T iem (min)
mV
�LCMS Chromatogram
�HPLC Chromatogram• HPLC
- Column :C18 5µµµµm хххх 25cm хххх 4.6mm
- Detector : UV 206nm
- Flow rate : 1.4mL/min
- Injection volume : 20µµµµL
- Mobile phase : Gradient
80
45
45
80
Water (%)
20
55
55
20
00
30
35
45
Acetonitrile (%)Time (min)
� Asiaticoside distribution percentage
of each tissue of Centella asiatica
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
the whole plant cultures of Centella asiatica (L/) Urban by elicitors, Physiology and
Biochemistry (2004), 23:339-344
� Conventional liquid solvent extraction
Experiment methods
Analysis
- Centella asiatica 3g + 100mL solvent (MeOH, EtOH, Water)
- Temperature: Room temperature
- Stir velocity : 1200rpm
- Time : 24hr
� Heat reflux extraction
- Centella asiatica 3g + 100mL solvent
- Temperature : MeOH(64 oC), EtOH(78 oC)
Water (100 oC)
- Time :5 hr
� Soxhlet extraction
�SCCO2 extraction
P1 T
T
3
6
5
10
11
12
12
1314
15
1. Extractor
2. Jacket
3. Pre-Heater
4. Pressure Gauge
5. Thermocouple
6. Back Pressure Regulator
7. Separator fraction
8. Activate Carbon Filter 1st
9. Activate Carbon Filter 2nd
10. Condensor
11. CO2 Pump
12. Circulator
13. CO2 Feed Tank
14. Level Cell
15. CO2 Cylinder
16. Co-solvent vessel
9
T P
P
T
7
8
T P
2
16
P
---345.230050
Time
(hr)
Static
Flow rate
(mL/min)
Co-solvent
Co- solvent
(%)
Time
(hr)
Dynamic
Flow rate
(mL /min)
CO2
P
(bar)
T
(oC)
� Pure SCCO2 extraction
26.1EtOH(65): water(35) =10%161.340080
24.52EtOH(65): water(35) =10%145.240050
Time
(hr)
Static
Flow rate
(mL/min)
Co-solvent
Co- solvent
(%)
Time
(hr)
Dynamic
Flow rate
(mL /min)
CO2
P
(bar)
T
(oC)
30 min 180 min 30 min 180 min
80 oC 50 oC
Results
Conclusions
- Centella asiatica 3g + 100mL solvent
( MeOH, EtOH, Water mixture)
- Temperature : Boiling point
- Time :5 hr
� SCCO2 + co solvent extraction
400bar, dynamic time : 3 h, static time : 1h
Time (min)
20 40 60 80 100 120 140 160 180 200
Asiaticoside (mg / g)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
50 oC
80 oC
(a)
Solvent o f Extraction
MeOH
EtOH
Water
MeOH
EtOH
Water
MeOH
EtOH
Water
EtOH
(25):Water(75)
EtOH
(50):Water(50)
EtOH
(75):Water(25)
Asiaticoside (mg) / Centella asiatica (g)
0
2
4
6
8
10
12
14
16
18
20
Conventional
Extraction
(T im e : 24 hr)
Heat o f re flux
Extraction
(T im e : 5hr) Sxohlet
Extraction
(T im e : 5 hr)
�The best extraction method of asiaticoside is heat of reflux extraction using ethanol.
� 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.
Asiatic acid
Asiaticoside
• LC-MS
- Column :C18 5 µµµµm хххх 25 cm хххх 4.6 mm
- Ionization mode : API/ES negative & positive mode
- Drying gas flow : 20 mL/min
- Drying gas temperature : 250 oC
- Capillary voltage : 4.5 kv
Supercritical Fluid Lab.Supercritical Fluid Lab.
