Extraction of Extraction of Asiaticoside Asiaticoside from from Centella Centella asiatica asiatica : : Effects of solvents and extraction methods Effects of solvents and extraction methods Wan Wan - - Joo Joo Kim Kim 1) 1) , , Jae Jae - - Duck Kim Duck Kim 1)* 1)* , , Bambang Bambang Veriansyah Veriansyah 1) 1) , , Jaehoon Jaehoon Kim Kim 1) 1) Seung Seung - - Geun Geun Oh Oh 2) 2) and Raymond R Tjandrawinata and Raymond R Tjandrawinata 3) 3) 1) Energy and Environment Research Division, Korea Institute of 1) Energy and Environment Research Division, Korea Institute of Science and Technology (KIST), Seoul, 136 Science and Technology (KIST), Seoul, 136- 791, Korea 791, Korea 2) Division of Chemical & Bioengineering, 2) Division of Chemical & Bioengineering, Hanyang Hanyang University, Seoul, 133 University, Seoul, 133- 791, Korea 791, Korea 3) PT 3) PT Dexa Dexa Medica Medica Graha Graha Elnusa Elnusa Building 5th Floor Building 5th Floor Jl Jl . TB . TB Simatupang Simatupang Kav Kav . 1B Jakarta 12560 INDONESIA . 1B Jakarta 12560 INDONESIA Centella asiatica Constituents Constituents Main 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) - SCCO 2 (Supercritical carbon dioxide) extraction centella extract # 118-151 RT: 2.14-2.76 AV: 34 NL: 2.32E7 T: + 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 871.41 975.75 521.74 887.29 991.49 593.22 956.71 486.74 216.69 390.79 1221.92 634.98 903.44 298.26 1322.00 813.19 683.91 1046.91 1121.98 533.12 1479.65 1369.76 758.81 1262.86 Asiaticoside Madecasoside Madecassic acid 505.8 8 Asiatic acid CH 3 OH OH HOCH 2 CH 3 CH 3 CH 3 CH 3 OH O CH 3 HO HO HOH 2 C C O H OH H H OH OH H H CH 2 O H OH H H OH H H CH 2 OH O OH OH H H H H OH CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 (10) 190 390 590 790 990 1190 0 5 10 15 20 25 30 35 40 45 50 Time(min) mV Centella Asiatica 0 20 40 60 80 100 120 140 160 180 200 0 5 10 15 20 25 30 35 40 45 50 Tiem (min) mV LCMS Chromatogram HPLC Chromatogram • HPLC - Column :C 18 5μm х 25cm х 4.6mm - Detector : UV 206nm - Flow rate : 1.4mL/min - Injection volume : 20μL - 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 o C), EtOH(78 o C) Water (100 o C) - Time :5 hr Soxhlet extraction SCCO 2 extraction P 1 T T 3 6 5 10 11 12 12 13 14 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. CO 2 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 - - - 3 45.2 300 50 Time (hr) Static Flow rate (mL/min) Co-solvent Co- solvent (%) Time (hr) Dynamic Flow rate (mL /min) CO 2 P (bar) T ( o C) Pure SCCO 2 extraction 2 6.1 EtOH(65): water(35) =10% 1 61.3 400 80 2 4.52 EtOH(65): water(35) =10% 1 45.2 400 50 Time (hr) Static Flow rate (mL/min) Co-solvent Co- solvent (%) Time (hr) Dynamic Flow rate (mL /min) CO 2 P (bar) T ( o C) 30 min 180 min 30 min 180 min 80 o C 50 o C Results Conclusions - Centella asiatica 3g + 100mL solvent ( MeOH, EtOH, Water mixture) - Temperature : Boiling point - Time :5 hr SCCO 2 + 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 o C 80 o C (a) Solvent of Extraction MeOH EtOH Water MeOH EtOH Water MeOH EtOH Water Et OH(25):Water(75) E t O H(50): Water(50) Et OH(75):Water(25) Asiaticoside (mg) / Centella asiatica ( g) 0 2 4 6 8 10 12 14 16 18 20 Conventional Extraction (Time : 24 hr) Heat of reflux Extraction (Time : 5hr) Sxohlet Extraction (Time : 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 SCCO 2 extraction can be extracted more effective than pure SCCO 2 about asiaticoside. Asiatic acid Asiaticoside • LC-MS - Column :C 18 5 μm х 25 cm х 4.6 mm - Ionization mode : API/ES negative & positive mode - Drying gas flow : 20 mL/min - Drying gas temperature : 250 o C - Capillary voltage : 4.5 kv Supercritical Fluid Lab. Supercritical Fluid Lab.