| Abstract |
- Douglas-fir bark was extracted with n-hexane. A light-colored
"wax-like" solid was recovered from the extract by evaporation of
the solvent. Chemically intact sterol esters and ferulic acid esters
were isolated from the "wax" without saponification or degradation
as had been necessary in former investigations. The characterization
of the intact esters provides an improved understanding of the
characteristics of the extracted "wax." Previous investigations had
been limited to an identification of compounds from saponified and
chemically fragmented "wax."
The intact, undegraded sterol and ferulic acid esters were
isolated by subjecting the n-hexane-soluble materials to column
chromatography using Silica Gel G as stationary phase and chloroform-n-hexane (3:1 v/v) as developing solvent. The effluents were collected
in different fractions and numbered from 1 to 75. Fractions 1, 2, 4, 6, 9, and 11 were tested by thin-layer
chromatography using diethyl ether-n-hexane (1:4 v/v) and chloroform-carbon
tetrachloride (6:1 v/v) as the solvent systems. The chromatograms
showed two spots (R[subscript]f 0.96 and 0.76). Positive results for
Liebermann-Burchard tests were found in all of the above fractions
except fraction 11, with the most prominent color reaction in fraction
2. Thin-layer chromatographic and gas-liquid chromatographic
results showed no free sterols in these fractions and the materials
were thus considered to be sterol esters. Fraction 2 was then subjected
to saponification since it showed the strongest Liebermann-
Burchard test.
The unsaponifiable fractions were tested by thin-layer chromatography
and gas-liquid chromatography. The results showed the
existence of campesterol and [Beta]-sitosterol. Their presence was
further confirmed by combined gas-liquid chromatographic-mass
spectrometry analyses.
The saponifiable and diethyl ether-soluble fraction was analyzed
by gas-liquid chromatography and combined gas-liquid chromatographic-mass
spectrometry analyses. The results showed the existence of n-tridecanoic
acid, n-hexadecanoic acid, n-heptadecanoic acid, cis-9-octadecenoic acid, n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic
acid and the possible existence of n-nonadecanoic acid.
Any or all of these fatty acids can form esters with the two sterols. The presence of sterol esters, especially esters of odd-numbered
fatty acids, has not been previously reported in Douglas-fir bark.
Fractions 13 to 17 on the column chromatograph showed a dark
blue coloration when exposed to ultraviolet light. The materials in
these fractions were collected and purified by recrystallization and
thin-layer chromatography. Infrared spectra of the purified material
showed it to be an ester. A saponification reaction was then conducted
to study the acid part and the alcohol part of the ester. The alcohol
portion was found to be a mixture of behenyl alcohol and lignoceryl
alcohol by studies of melting point, gas chromatography, and infrared
and mass spectrometry.
The acid part was proven to be ferulic acid by melting point
test, infrared spectroscopy, field desorption mass spectrometry, and
nuclear magnetic resonance spectroscopy. The infrared spectrum of
the ester showed the presence of hydroxyl groups. Therefore, the
ester linkage was between the carboxylic acid group of ferulic acid
and the hydroxyl group of the two fatty alcohols. The ferulic acid
ester was thus different from the ester found in white fir bark. The
present work is the first report to show that ferulic acid does not
exist in the free form in n-hexane "wax" as had been suggested
earlier. It exists as an ester.
The n-hexane-soluble materials were also shown to contain
some terpenes, alcohols and other easily volatilized components. To
avoid heavy losses, the volatile materials were collected by steam
distillation of the bark rather than to attempt to separate them from
the n-hexane-soluble fraction. Combined gas-liquid chromatographic-mass
spectrometry analyses showed the major volatile components to
be furfural, terpinene-4-ol, [alpha]-terpineol, guaiacol, 2, 5-dimethyl-3-
acetylfuran, and [Beta]-cyclocitral. The presence of [alpha]-pinene, [Beta]-pinene,
limonene, 1, 5-p-menthadien-7-ol, {Beta]-citronellol, and gerniol was also
tentatively identified. The presence of [alpha]-terpineol is of interest to
forest entomologists because it is known to be an insect attractant
for bark beetles. The present work is the first report concerning the
presence of furfural, terpinene-4-ol, [alpha]-terpineol, guaiacol, 2, 5-dimethyl-3-acetylfuran, and [Beta]-cyclocitral in Douglas-fir bark.
|
|---|
