Seminarieuppgifter till 2015.10.12 "extraktion & isolering"
Det har väl inte undgått någon på kursen att upptäckten av artemisinin
nyligen belönades med Nobelpris, tillsammans med upptäckten av
avermectin. Historien bakom dessa substanser speglar två
strategier för läkemedelsupptäckt inom farmakognosin:
genom att utnyttja etnofarmakologi och traditonell medicin
som i fallet med artemisinin, och ett mer brett gränslöst sökande
som i fallet med avermectin. Den första uppgiften till seminariet
handlar om isoleringen av en av dessa substanser, artemisinin.
Det första som publicerades till allmän kännedom om själva isoleringen av artemisinin
kom däremot inte från upptäckarna – den forskargrupp som leddes av Youyou Tu under
namnet “Qinghaosu Antimalaria Coordinating Research Group” arbetade i hemlighet
under lång tid.
Klippen nedan (i något modifierade former för att passa seminariet) är hämtade från
“Isolation of artemisinin (qinghaosu) from Artemisia annua growing in the United States,
av Klayman DL, et al, i Journal of Natural Products, 1984: 47(4):715-7.
“Because artemisinin has a structure totally different from existing antimalarials, we
were prompted to ascertain to what extent the A. annua found in the United States
contains this constituent. Thus, the plant, which grows nearby as a weed, was
collected and air-dried. Several low boiling solvents (e.g., CH2C12, CHCI3, Et2O,
Me2CO) extracted artemisinin readily; however, petroleum ether (30-60°) was
most selective and, therefore, considered to be the solvent of choice. Treament of
the petroleum ether extract with CH3CN removed much of the accompanying
waxes, and further fractionation of the extract on silica gel gave artemisinin.
Whereas the dried leaves and/or flowers yielded artemisinin, the stems of the plant
were found to be devoid of the compound.
Chinese workers reported that a study of 30 other Artemisia species (not identified)
failed to uncover any with antimalarial properties. We examined ten species that
grow in the United States by the extraction procedure outlined above and similarly
did not find any containing artemisinin. In view of the earlier reports of failure to
detect antimalarial activity in EtOH extracts of A. annua, we examined the stability
of artemisinin boiled in this solvent for 48 h. It is estimated by TLC that ca. 20%
of the sample was destroyed by such treatment, as indicated by the appearance of a
new spot at the origin. In addition, there was a broadening of the carbonyl peak at
1745 cm-'. Treatment of artemisinin with iso-PrOH at reflux temperature for 48 h,
however, led to the recovery of unchanged starting material.”
I material och metod-delen står vidare:
“EXTRACTION AND FRACTIONATION. Air dried leaves of A. annua (200 g)
collected in early August 1983, were extracted with boiling petroleum ether (bp 3060°) for 48 h. Removal of the solvent in vacuo gave a dark brown syrup that was
dissolved in 20 ml of CHCI3, and to this solution was added 180 ml of CH3CN.
The insoluble material was removed, and the filtrate was evaporated under reduced
pressure to give 4.5 g of gummy residue.
CHROMATOGRAPHIC SEPARATION. The residue was chromatographed on
200 g of 70-230 mesh silica gel using 7.5% EtOAc in CHCI3, as the eluant.
Fractionation was begun after passage of 200 ml of eluant and the aliquots were
monitored by TLC (silica gel plates, 7.5% EtOAc in CHCI3; detection of
artemisinin by iodine vapor; Rf=O.66). Artemisinin appeared after collection of
about 300 ml of eluant and was obtained (0.12 g) as fine white crystals, mp 153154° (lit. mp 156-157°), after recrystallization from cyclohexane. The identity of
artemisinin with an authentic sample of was confirmed by comparative melting
point, superimposable IR spectrum. The proton and carbon NMR spectra were
identical to those reported. Mass spectrum m/z 283 (M+1); calcd (C15H22O5,)
282.
Uppgift 1.
a) Sammanfatta isoleringen schematiskt, och förklara kort vilken betydelse de olika
stegen fram till ett rent, och identifierat, artemisinin har.
b) Beräkna utbytet av artemisinin.
Nästa uppgift är också saxad ur Journal of Natural Products, och artikeln “Coumarins
from Galipea panamensis and Their Activity against Leishmania panamensis” av Arango et al.,
(2010, 73, 1012-1014).
I abstraktet kan man läsa följande,
“Two new coumarin compounds (1 and 2), phebalosin (3), its derived artifact
murralongin (4), and murrangatin acetonide (5) were isolated from the leaves of
Galipea panamensis. The structures of 1 and 2 were assigned as 7-{[(2R*)-3,3dimethyloxiran-2-yl]methoxy}-8-[(2R*,3R*)-3-isopropenyloxiran-2-yl]-2Hchromen-2-one and 7-methoxy-8-(4-methyl-3-furyl)-2H-chromen-2-one,
respectively, on the basis of their spectroscopic data (primarily NMR and MS).
Compounds 1−3 were tested against axenic amastigote forms of Leishmania
panamensis and displayed 50% effective concentrations (EC ) of 9.9, 10.5, and 14.1
50
µg/mL, respectively. These three compounds also displayed cytotoxicity (IC ) at
50
concentrations of 9.7, 33.0, and 20.7 µg/mL, respectively, on human promonocytic
U-937 cells.”
och detaljerna för isoleringen specificeras i Experimental Section:
“Extraction and Isolation. Powdered leaves (1.0 kg) of G. panamensis were extracted
successively with petroleum ether, EtOAc, and MeOH (10 L each) in a percolator
at room temperature and concentrated in vacuo to give the corresponding extract
(8, 34, and 44 g, respectively). The ethyl acetate extract was subjected to silica gel
column chromatography (5 × 80 cm) eluting with a step gradient of nhexane−ethyl acetate (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80,
10:90, 0:100, each 500 mL), to obtain 10 fractions (F1−F10) collected on the basis
of their TLC profiles. Fractions F4 and F5 were recognized as the most interesting
ones, due to the appearance of blue spots after spraying with anisaldehyde reagent.
Compounds 2 (30 mg) and 3 (38 mg) were isolated from F4, and compounds 1 (35
mg) and 5 (12 mg) from F5, by preparative TLC using CH Cl −EtOAc (4:1),
2
2
except for compound 2, for which an n-hexane−ethyl acetate (4:1) mixture was
employed. Compound 4 appeared during purification of phebalosin (1).”
Uppgift 2.
A) Beskriv isoleringen med ett isoleringsschema
B) En perkolator användes för extraktionen: vad innebär den extraktionsmetoden
och vad har den för fördelar?
C) Skulle du vilja beskriva isoleringen av föreningarna 1 och 2 som bioaktivitetsstyrd (bioassay guided)? Motivera ditt svar.
Mer läsning/inspiration? Förutom kursboken, se länken till boken “Natural Products Isolation”
på kursens hemsida; kapitlet “Extraction of Plant Secondary Metabolites” av William P. Jones
och A. Douglas Kinghorn rekommenderas särskilt.