Authors: Nguyễn Công Toại , Nguyễn Khánh Hạ
Company/Faculty/School: HUS High School for Gifted Students
This invention is filed patent. The invention relates to a process of extracting bioactive eupatoriopicrin from the leaves of Eupatorium japonicum Thunb. with methanol at room temperature, eliminating less polar compounds by n-hexane, extracting eupatoriopicrin into a dichloromethane phase, isolating eupatoriopicrin as an amorphous powder, and purifying the said compound in its crystalline form. The natural bioactive eupatoriopicrin extracted from the material is safe and can be used in the preparation of drugs or pharmaceutical preparation to treat human lung cancer, fibrosarcoma, liver cancer, and breast cancer.
Novelty and Innovation, Utility
1. A process for the extraction and purification of eupatoriopicrin from the leaves of Eupatorium japonicum Thunb. comprising the steps of:
a) Preparation of the dried leaves of Eupatorium japonicum to meet the standards of materials: washing the fresh leaves, air-drying the leaves in the shadow, then oven-drying at 45 oC for one day;
b) Grinding the dried leaves into powder to increase the contacting areas between the material and extraction solvents to optimize the extraction yield of organic compounds from the leaves;
c) Extracting the dried leaf powder in methanol at room temperature for 3 days; the process is repeated 3 times;
d) The methanol extracts are combined and evaporated to remove methanol under reduced pressure at 50 oC to yield a methanol extract; diluting the said extract with distilled water and extracting compounds according to their polarities between a water phase and organic solvents of increasing polarities;
e) Partitioning less polar compounds into an n-hexane phase (3 times), drying the phase with Na2SO4, and evaporating solvent under reduced pressure at 50 oC to yield an n-hexane extract;
f) Extracting the remaining water phase with dichloromethane (3 times), drying with Na2SO4, and evaporating solvent under reduced pressure at 40 oC to yield a dichloromethane extract;
g) The said dichloromethane extract containing eupatoriopicrin is impregnated on silica gel (Merck 63-200 μm) and separated by column chromatography over silica gel (Merck 63-200 μm), eluting with CH2Cl2 and CH2Cl2–acetone 95:1, 35:1, 19:1 (v/v) solvent systems to yield 2 pooled fractions. The pooled fraction 2 containing eupatoriopicrin is diluted in a minimal amount of dichloromethane and impregnated on silica gel (Merck, 63-200 μm), then is loaded onto a silica gel (Merck, 40-63 μm) column. The column chromatography is eluted with n-hexan-EtOAc 3:1, 2:1, 1:1 (v/v) to yield 4 pooled fractions. Eupatoriopicrin is crystallized from the pooled fraction 3 from n-hexane-EtOAc solvent system used for the column chromatography.
h) In each repeated batch the extraction and separation processes are controled by thin-layer chromatography analysis (TLC, silica gel Merck, DC-Alufolien 60 F254, solvent system CH2Cl2-acetone 6:1, v/v) with eupatoriopicrin as the reference compound.
2. The bioactive eupatoriopicrin obtained according to the process in claim 1 used in the preparation of drugs and pharmaceutical preparations to treat human lung cancer, fibrosarcoma, liver cancer, and breast cancer