GLYCOSIDES IN Viola Tricolor L.

CHAPTER 3: MATERIAL AND METHODS
acetate,and(iv)70%methanol. Theextractswereevaporatedtodrynessunder
reduced pressure, andre-dissolved in chloroform or100%methanol.
Method 2 5.0g dried and freshly powdered heartsease herb was sonicated
two times with 50mL chloroform for 25 minutes in an ultrasonic bath at 30 ◦ C.
The chloroform extract, referred to as Fraction A (Fig. 4.1) was evaporated to
dryness under reduced pressure at 30 ◦ C. The plant residue was dried at room
temperature and re-extracted two times with 40mL methanol for 15 minutes
in an ultrasonic bath at 30 ◦ C. The methanol extract referred to as Fraction B
(Fig. 4.1)wasevaporated todryness underreduced pressure at40 ◦ C.
Method 3 1.25g dried and freshly powdered heartsease plant material was
subject of continuous Soxhlet extraction with 100mLmethanol for 9h(exhaus-
tiveextraction). Theextractwasevaporatedtodrynessunderreducedpressure
at 55 ◦ C, weighed and re-dissolved in 25mL methanol (stock solution). 1.5mL
0.085% phosphoric acid was added to 3.5mL of the extract (diluted stock solu-
tion)andfilteredonaMINIsartRC-15,0.20µmmembranesyringefilter(Sigma-
Aldrich). The filtered solution wasreadyto beanalyzed.
Method 4 0.50g air-dried plant material was sonicated with 20mL 70% me-
thanol for 20 minutes in an ultrasonic bath at room temperature. The filtered
extract was evaporated to dryness in vacuum at 60 ◦ C. The dry residue was
re-dissolved in 1.5 mL 70% methanol and separated by solid-phase extraction
(Sec. 3.4).
3.4 Solid-phaseextraction
SamplesforSPEwerepreparedsoastobeequivalentof20%methanol. TheSPE
cartridge(SupelcleanLC-18,500mg/3mL,Sigma-Aldrich)wasactivatedwith
3mLmethanolfollowedby3mL2%aceticacid. Aftersampleintroduction, the
cartridge was washed with 1.5mL of 70% methanol. The loading and washing
solvents were collected andcombined fordownstream analysis.
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