Please do not adjust margins
Green Chemistry
Page 3 of 10
Journal Name
DOI: 10.1039/C6GC02641A
ARTICLE
3H), 1.05 (m, 3H), 1.18-1.28 (m, 4H), 1.39 (m, 5H), 1.51-1.70 and 19 µl t-BuOH, 93 µl water and 1236 µl HOAc were added
(m, 12H), 1.85 (m, 1H), 1.94 (m, 2H), 2.38 (m, 1H), 3.19 (dd, J = and the solution was stirred at room temperature for 6 h.
1
1.5, 4.8, 1H), 3.33 (dd, J = 10.9, 1.3, 1H), 3.80 (dd, J = 10.8, Pinnick reagents 2-methyl-2-butene (47 µl, 33 mg, 0.47 mmol,
1
3
1
.6, 1H), 4.58 (s, 1H), 4.68 (s, 1H). C-NMR (150 MHz, CDCl
= 15.1, 15.7, 16.3, 16.5, 18.6, 19.4, 21.2, 25.5, 27.4, 27.7, NaH
8.3, 29.5, 30.1, 34.3, 34.6, 37.5, 37.6, 39.0, 39.2, 41.3, 43.1, change to brown was observed while a white solid was
3
2
): 4.6 eq.), NaClO (30 mg of 80% purity, 0.27 mmol, 2.6 eq.) and
δ
2
4
C
2
PO (32 mg, 0.27 mmol, 2.6 eq.) were added and a color
4
8.1 (2C), 49.1, 50.7, 55.6, 60.9, 79.3, 110.1, 150.8.
precipitating. The solution was quenched with saturated
sodium thiosulfate solution and extracted with EtOAc three
1
4
Analytical data was in accordance with literature values.
times, washed with sat. NaHCO
Isolation of betulin via microwave extraction. In a 10 ml Na SO . The solution was filtered and evaporated to dryness.
microwave vial birch bark (101.0 mg) and 900 mg 40% The off-white residue was purified using column
4444]OH solution were stirred for 15 min at 100 °C under MW chromatography (3 g SiO , PE-EtOAc = 20:1-2:1) to yield
irradiation. Water (2 ml) and 3 ml of n-BuOAc were added and betulinic acid ( ) in 37.5 mg corresponding to 18 wt% yield as
the mixture was again subjected to MW irradiation. The colorless crystals with a purity of > 95% according to H NMR.
3
solution and dried over
2
4
[P
2
1
1
1
4
organic layer was separated and concentrated under reduced Analytical data was in accordance with literature.
pressure.
Isolation of betulinic acid via crystallization. The procedure
After purification via column chromatography (7 g SiO
2
,
was performed as described above using 507.5 mg birch bark,
PE:EtOAc = 20:1-2:1) 25.4 mg betulin were obtained as 4.5 g 40% [P4444]OH solution, 10 ml water and 15 ml BuOAc.
colorless crystals in 25 wt.% yield corresponding to birch bark.
Oxidation reagents were calculated on 130 mg betulin (
2). The
crude product was crystallized from a mixture of
Oxidation of betulin to betulinic acid
methanol/water to yield betulinic acid (111 mg, corresponding
to 21.9 wt.% yield) as colorless crystals with a purity of >90%
according to NMR. A second fraction of betulinic acid (36.9 mg,
corresponding to 7.0 wt.% yield) was obtained with a purity
Betulin (2, 99.5 mg, 0.2247 mmol), TEMPO (7.0 mg, 0.04495
mmol, 0.2 eq.) and BAIB (217 mg, 0.6742 mmol, 3 eq.) were
dissolved in 10 ml n-BuOAc, 0.2 ml water and 42 µl t-BuOH.
After 6 h stirring at room temperature TLC showed full
conversion. To the clear yellow solution 0.2 ml water, 2-
<
90% according to NMR.
methyl-2-butene (72 µl, 1.03 mmol, 4.6 eq.), NaH
2
PO
(66 mg with 80% purity,
.584 mmol, 2.6 eq.) were added and a colour change to Biphasic extraction of betulin at room temperature
4
(70 mg,
Results and Discussion
0.584 mmol, 2.6 eq.) and NaClO
2
0
brown was observed while a white solid was precipitating. The
solution was quenched with saturated sodium thiosulfate
solution and extracted with EtOAc three times, washed with
The extraction of betulin (
performed using conventional Soxhlet extraction with
number of solvents such as dichoromethane, chloroform,
2) from birch bark is typically
a
2
4,25,26
saturated NaHCO
3 2 4
solution and dried over Na SO . The
ethanol or methanol.
However, these conventional
solution was filtered and concentrated under reduced
pressure. The off-white residue was crystallized from MeOH to
extraction strategies typically require large solvent volumes
and suffer from long extraction times at elevated
temperatures, resulting in high time and energy consumption.
As for betulinic acid, the purity of the raw extracts is typically
low, and additional purification steps are usually required.
As a consequence, efforts to reduce the exhaustive use of
undesirable solvents as extraction media for betulin have been
made, and recent trends include the use of bio-derived
obtain betulinic acid in 89% yield as colourless crystals.
1
m.p.: 284.5-285.5 °C. H-NMR (400 MHz, CDCl
3
): δ
H
= 0.67 (m,
1
H), 0.75 (s, 3H), 0.8 (s, 3H), 0.93 (s, 3H), 0.96 (s, 3H), 0.97 (s,
H), 1.18- 1.38-1.69 (m, 15H), 1.69 (s, 3H), 1.97 (m, 2H), 2.19
3
(
m, 1H)1.27 (m, 5H), 2.26 (m, 1H), 3.00 (m, 1H), 3.19 (dd, J =
1
3
1
1.3, 5.0, 1H), 4.60 (s, 1H), 4.74 (s, 1H). C-NMR (100 MHz,
): δ = 15.1, 15.7, 16.4, 16.5, 18.6, 19.7, 21.2, 25.8, 27.8,
8.3, 30.1, 30.9, 32.5, 34.7, 37.4, 37.6, 38.7, 39.1, 39.2, 41.0,
2.8, 47.2, 49.6, 50.9, 55.7, 56.6, 79.4, 110.1, 150.8, 180.2.
2
7
10
CDCl
3
C
solvents such as limonene,
pressurized solvent
and
2
supercritical fluid extraction or ionic liquid-based extraction
8
2
2
9
4
strategies. In a previous paper, we reported the microwave-
assisted extraction of betulin ( ) using several hydrophilic and
1
4
Analytical data was in accordance with literature.
2
hydrophobic ionic liquids. Ionic liquids as solvents for the
extraction of value-added compounds may result in increased
yields compared to conventional solvents through
Streamlined extraction and oxidation process
Isolation of betulinic acid via chromatography. In a 20 ml
3
0,31
fractionation of the lignocellulosic matrix.
In case of
screw-cap vial 200.2 mg ground birch bark and 1800 mg of a
betulin, the best extraction yield was obtained after
dissolution of birch bark in the ionic liquid 1-ethyl-3-
2
40% [P4444]OH solution in H O were stirred for 1 h at room
temperature. Water (2 ml) and n-BuOAc (3 ml) were added
and the mixture was stirred vigorously for 1 h at room
temperature. The organic layer was separated and filtered
2
methylimidazolium acetate ([C mim]OAc) at 100 °C for 15 min.
An isolation strategy relying on the stepwise precipitation of
biopolymers with ethanol followed by the addition of water to
crystallize betulin was developed and allowed isolating betulin
in good yields and purity.
over a Pasteur pipette filled with SiO
extract was concentrated to obtain 10 mg betulin/ml. TEMPO
3 mg, 0.021 mmol, 0.2 eq.), BAIB (99.5 mg, 0.31 mmol, 3 eq.)
2 2 4
and Na SO . The crude
(
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins