American Journal of Analytical Chemistry, 2010, 1, 31-33
doi:10.4236/ajac.2010.11004 Published Online May 2010 (http://www.SciRP.org/journal/ajac)
31
Steroids from the Whole Plants of Leucaena
Leucocephala
Chung-Yi Chen*, Yau-Der Wang
School of Medical and Heath Science, Fooyin University, Kaohuing , Taiwan, China
Received January 10, 2009; revised February 21, 2009; accepted February 23, 2009
Abstract
Four steroids, 5α,8α-epidioxy-(24ξ)-ergosta-6,22-dien-3β-ol (1), β-sitosterol (2), β-sitostenone (3), and stig-
mastenone (4), along with 10 known compounds were isolated from the whole plants of Leucaena leuco-
cephala (Leguminosae). 1, Lupeol (5), 1,3-dipalmitoyl-2-oleoylglycerol (6), methylparabene (8) and isova-
nillic acid (9) were found for the first time from the species. The structure of these compounds were charac-
terized and identified by spectra analyses.
Keywords: Steroids, Leucaena Leucocephala, Leguminosae
1. Introduction
Leucaena leucocephala (Leguminosae) is a small, legu-
minous and native to tropical America, now widely dis-
tributed in southern Asia and neighboring islands [1].
Previous studies have show that gallocatechin, epigallo-
catechin, catechin and epicatechin, extracted from the
roots of L. leucocephala was found to exhibit the nitrifi-
cation inhibition bioassay against the bacterium Nitro-
somonas europaea [2]. L. leucocephala was chosen for
further phytochemical investigation. The MeOH extract
of its plants were subjected to solvent partitioning and
chromatographic separation to afford 14 pure substances.
The chemical constituents in the plants of L. leuco-
cephala were separated with column chromatography.
Investigation on the MeOH extract of the plants has
led to the isolation of 14 compounds, four steroids: 5α,
8α-epidioxy-(24ξ)-ergosta-6,22-dien-3β-ol (1) (Figure 1)
[3], β-sitosterol (2) (Figure 2) [4], β-sitostenone (3)
(Figure 3) and stigmastenone (4) (Figure 4) [5] ; one
terpenoid: lupeol (5) [6]; one glyceride: 1,3-dipalmitoyl-
2-oleoylglycerol (6) [7]; one alkanoid: linoleic acid (7)
[8]; two benzenoids: methylparabene (8) [9] and isova-
nillic acid (9) [10]; and five chlorophylls: pheophytin-a
(10) [11], pheophorbide a methyl ester (11) [12],
methyl-132- hydroxy-(132-S)-pheophorbide-b (12) [13],
132-hydroxy-(132-S)-pheophytin-a (13) [14] and aristo-
phyll-C (14) [15]. These compounds were obtained and
characterized by the comparison of their physical and
spectral data (UV, IR, NMR and MS) with values ob-
tained in the literature. Among them, 1, 5, 6, 8 and 9
were found for the first time from the species.
The specimen of L. leucocephala was collected from
Pingtung County, Taiwan in December, 2008. A voucher
specimen was characterized by Dr. Jin-Cherng Huang of
Department of Forest Products Science and Furniture
Engineering, National Chiayi University, Chiayi, Taiwan
and deposited in the School of Medical and Health Sci-
ences, Fooyin University, Kaohsiung County, Taiwan.
The air-dried seeds of L. leucocephala (5.0 kg) were ex-
tracted with MeOH (80 L × 6) at room temperature and
the MeOH extract (162.5 g) was obtained upon concen-
tration under reduced pressure. The MeOH extract was
chromatographed over silica gel (800 g, 70-230 mesh)
using n-hexane/acetone as eluent to produce 5 fractions.
Part of fraction 1 (6.11 g) was subjected to Si gel chro-
matography by eluting with n-hexane/acetone (40:1),
HO
O
O
Figure 1. Chemical structure of 5α,8α-epidioxy-(24ξ)-ergosta-
6,22-dien-3-ol (1).

C. Y. CHEN ET AL. 32
HO
Figure 2. Chemical structure of β-sitosterol (2).
O
Figure 3. Chemical structure of β-sitostenone (3).
O
Figure 4. Chemical structure of stigmastenone (4).
enriched with acetone to furnish 10 fractions (1-1 to
1-10). Fraction 1-1 (1.72 g) was re-subjected to Si gel
chromatography, eluting with n-hexane/acetone (80:1) to
obtain linoleic acid (7) (58 mg, 0.0357%). Part of frac-
tion 3 (6.94 g) was subjected to Si gel chromatography
by eluting with n-hexane/acetone (50:1) to obtain
β-sitosterol (2) (15 mg, 0.0092%). Part of fraction 4
(6.77 g) was subjected to Si gel chromatography by
eluting with n-hexane/acetone (8:1), then enriched with
acetone to furnish 7 fractions (4-1 to 4-7). Fraction 4-5
(0.62 g) was further purified by another silica gel column
using n-hexane/acetone to obtain methylparabene (8)
(8 mg, 0.0049%) and isovanillic acid (9) (12 mg, 0.0074%).
The air-dried brown beans of L. leucocephala (5.0 kg)
were extracted with MeOH (80 L × 6) at room tempera-
ture and the MeOH extract (171.5 g) was obtained upon
concentration under reduced pressure. The MeOH extract
was chromatographed over silica gel using n-hexane/
acetone as eluent to produce 6 fractions. Part of fraction
1 (7.02 g) was subjected to Si gel chromatography by
eluting with n-hexane/acetone (40:1) to obtain
β-sitostenone (3) (6 mg, 0.0035%) and stigmastenone (4)
(4 mg, 0.0023%). Part of fraction 2 (8.49 g) was sub-
jected to Si gel chromatography by eluting with n-hexane/
acetone (10:1), then enriched with acetone to furnish 8
fractions (2-1 to 2-8). Fraction 2-3 (2.33 g) was re-sub-
jected to Si gel chromatography, eluting with n-hexane/
acetone (40:1) to obtain lupeol (5) (74 mg, 0.0431%).
Part of fraction 3 (7.76 g) was subjected to Si gel chro-
matography by eluting with n-hexane/acetone (40:1) to
obtain 1, 3-dipalmitoyl-2-oleoylglycerol (6) (9 mg,
0.0052%). Part of fraction 6 (13.89 g) was subjected to
Si gel chromatography by eluting with n-hexane/acetone
(4:1), then enriched with acetone to obtain 5α,8α-epidioxy-
( 24ξ)-ergosta-6,22-dien-3β-ol (1) (43 mg, 0.0251%).
The air-dried leaves of L. leucocephala (5.0 kg) were
extracted with MeOH (80 L × 6) at room temperature
and the MeOH extract (159.5 g) was obtained upon con-
centration under reduced pressure. The MeOH extract
was chromatographed over silica gel using n-hexane/
acetone as eluent to produce 7 fractions. Part of fraction
1 (6.62 g) was subjected to Si gel chromatography by
eluting with n-hexane/acetone (50:1) to obtain pheo-
phytin-a (10) (6 mg, 0.0038%) . Part of fraction 3 (7.56 g)
was subjected to Si gel chromatography by eluting with
n-hexane/acetone (8:1), then enriched with acetone to
furnish 12 fractions (3-1 to 3-12). Fractions 3-7 (2.97 g)
was re-subjected to Si gel chromatography, eluting with
n-hexane/acetone (40:1) to obtain 132-hydroxy-(132-S)-
pheophytin-a (13) (12 mg, 0.0075%) and methyl-132-
hydroxy-(13 2-S)-pheophorbide-b (12) (9 mg, 0.0056%).
Part of fraction 6 (17.15 g) was subjected to Si gel chro-
matography by eluting with n-hexane/acetone (4:1) to
obtain aristophyll-C (14 ) (4 mg, 0.0025%).
The air-dried green beans of L. leucocephala (5.0 kg)
were extracted with MeOH (80 L × 6) at room tempera-
ture and the MeOH extract (146.5 g) was obtained upon
concentration under reduced pressure. The MeOH extract
was chromatographed over silica gel using n-hexane/
acetone as eluent to produce 8 fractions. Part of fraction
4 (7.19 g) was subjected to Si gel chromatography by
eluting with n-hexane/acetone (8:1) to obtain pheophor-
bide a methyl ester (11) (8 mg, 0.0055%).
2. Acknowledgements
This investigation was supported by a grant from the

C. Y. CHEN ET AL. 33
National Science Council of Republic of China (NSC
97-2320-B-242-002-MY3).
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