British Journal of Pharmacology and Toxicology 2(5): 270-272, 2011
ISSN: 2044-2467
© Maxwell Scientific Organization, 2011
Submitted: September 08, 2011
Accepted: September 30, 2011
Published: November 25, 2011
Isolation of Steroids from Acetone Extract of Ficus iteophylla
1, 2
I.A. Abdulmalik, 2M.I. Sule, 2A.M. Musa, 3A.H. Yaro, 2M.I. Abdullahi,
1
M.F. Abdulkadir and 1H. Yusuf
1
Department of Applied Science, C.S.T. Kaduna Polytechnic-Nigeria
2
Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University,
Zaria -Nigeria
3
Department of Pharmacology, Faculty of Medicine, Bayero University Kano-Nigeria
Abstract: Two steroids were isolated from the leaves of Ficus iteophylla (Family: moraceae) a plant popularly
used in African traditional medicine to treat variety of illnesses. The leaf part of the plant was investigated
phytochemically using a standard procedure. Schematic fractionation of its ethanol extract by acetone and
methanol and subsequent column chromatography of the acetone fraction over silica gel G (60-120) mesh size
led to the isolation of 3$-cholest-5-ene-3, 23diol (1) and 24 ethyl cholest-5-ene- 3$-ol (2). The structure of
these two compounds was elucidated using 1H-NMR, 13C- NMR and DEPT analysis. To the best of our search
this is the first report on the isolation of these compounds from the leaves of Ficus iteophylla..
Key words: Ficus iteophylla; 1: H- NMR; 13: C- NMR, DEPT; 3$-cholest-5-ene-3; 23diol; 24 ethyl cholest-5ene-3$-ol
INTRODUCTION
solvent in both cases were removed at reduced pressure to
give 15 and 50 g of Petroleum Ether (PE) and ethanol
(EE), respectively. The Ethanol Extract (EE) was
successively partitioned with acetone followed by
methanol to give acetone extract coded EEAc (8 g) and
methanol extract coded EEM (16 g).
Ficus iteophylla belongs to family moraceae, The
bark is used to treat dysentery and rheumatic pain
(Burkill, 1997). The root has a wide usage for treating
paralysis, tuberculosis, epilepsies, convulsion, spasm and
pulmonary troubles (Burkill, 1997). The leaf part is
reported to have analgesic, anti-inflammatory activity
(Abdulmalik et al., 2011) and antibacterial activity
(Ahmadu et al., 2006). It is also reported to contain two
furanocoumarines (Ahmadu et al., 2004) and two
flavonoid glycosides (Ahmadu et al., 2006). In
continuation of investigation of bioactive metabolites
from Ficus iteophylla, we report herein the isolation and
identification of steroids from the leaf part of the plant.
Column chromatography of acetone extract from
ethanolic extract: The acetone extract from ethanol
extract (EEAc) was dissolved in small quantity of acetone
and was adsorbed on silica gel. It was allowed to dry and
ground into a fine powder. The fine powder was applied
over a well-packed silica gel G (60-120 mesh size)
column. The column was then eluted gradiently with
Hexane: Ethyl acetate mixture, with polarity increased
gradually. Eluents were collected as 30 mL fraction and
the progress of the separation was monitored by thin layer
chromatography, similar fractions were pooled together.
MATERIALS AND METHODS
Plant material: The plant samples were collected from
Ahmadu Bello University, Zaria Nigeria in the month of
March, 2006. It was authenticated by comparing with the
existing one by Mallam Musa Muhammad of the
Herbarium section of the Department of Biological
Sciences, Ahmadu Bello University, Zaria, Nigeria.
Instruments: The melting point was determined using
Gallemkemp capillary and melting point apparatus and
they were uncorrected. The 600 MHz H-NMR spectra
were recorded in CDCl3 with Teramethyl Silane (TMS) as
internal standard. The 13C NMR and DEPT were recorded
at 400MHz. The DEPT experiments were used to
determine the multiplicities of carbon atoms. Thin layer
chromatography (TLC) was performed on TLC Silica gel
60 F254 pre-coated (Merck). The spots were visualized by
spraying with 10% H2SO4 followed by heating at 100ºC
for 5 min.
Extraction procedure: The powdered leaves (850 g) of
the plant was exhaustively extracted with petroleum
ether(60-80ºC) using Soxhlet apparatus, the marc was
dried and extracted with ethanol (96%) in same way. The
Corresponding Author: I.A. Abdulmalik, Department of Applied Science, C.S.T. Kaduna Polytechnic-Nigeria, Tell.: +2348069451993
270
Br. J. Pharmacol. Toxicol., 2(5): 270-272, 2011
Table 1: 1H and 13C NMR chemical shift assignments of compounds 1
and 2 and DEPT spectrum of compound 2
1
2
-----------------------------------------------------------------------------13 a
1
13 3
C
Ha
C
DEPTb
Carbon 1Ha
1
38.9
1.0825
37.2665
CH2
2
37.7
1.5158
31.6687
CH2
3
3.59
79.0
3.5255
71.8239
CH
4
48.8
2.2860
42.3056
CH2
5
158.1
140.7623
C
6
5.48
116.8
5.3589
121.7271
CH
7
37.8
1.9951
31.9134
CH2
8
28.8
1.2534
23.0732
CH
9
29.9
1.1831
24.3059
CH
10
C
11
27.9
1.460
21.0852
CH2
12
41.3
1.1600
39.7822
CH2
13
C
14
63.2
0.9814
56.7756
CH
15
35.6
1.5857
29.1625
CH2
16
33.3
1.8573
28.2480
CH2
17
55.5
1.0915
50.1417
CH
18
0.85
18.8
0.6980
11.9817
CH3
19
1.01
17.4
1.0374
11.8602
CH3
20
38.8
1.3575
37.2561
CH
21
1.50
27.1
0.9292
19.0351
CH3
22
38.0
1.3229
33.9534
CH2
23
3.15
63.0
1.1718
26.0861
CH2
24
49.3
1.9198
45.8475
CH
25
35.7
1.6587
29.6965
CH
26
1.20
22.6
0.8265
18.7810
CH3
27
1.20
21.3
0.8265
18.7810
CH3
28
1.2288
23.0732
CH2
29
0.8530
19.8174
CH3
a
: Spectra recorded at 600MHz in CDCl3; b: Spectra recorded at 400MHz
in CDCl3
Fig. 1: Thin Layer Chromatography for compound 1 and 2)
HO HO Compound 1
proton signal (*H1.0 - *H1.8) attributed to resonance of
overlapping of methylenes and methines a characteristic
frame work of steroid (Yun-Song et al., 2006). It showed
a multiplet at *H 3.59 and 3.15. The signal at *H 3.59 and
3.15 revealed the presence of two hydroxyl group, these
signal were for proton on carbon a
djacent to alcohol.
The signal at *H 3.59 was ascribed to C-3 (XU and Zeng,
2000) and that at *H 3.15 was ascribed to C-23. The C-6
oleifinic proton appeared at *H 5.48. The 1H-NMR
showed vicinal coupling between C-3 methine proton and
C-2 methylene at *H 1.98 and *H 1.85 (López et al.,
2008), the apectrum further displaced signal at *H 0.85 (C18) and *H 1.01 (C-19) characteristic of 5-ene-3$-hydroxy
sterols, at *H 1.50 (C-21) and *H 1.20 (C-26 and C-27).
The 13C-NMR showed peaks at *C 11.8, *C 11.7, *C 19.0
corresponding to C-18, C-19 and C-21, respectively. The
C-3 and C-23 resonated at *C 79.0, *C 63.2, respectively.
C- 5 resonated at *C 158.1 while C-6 resonated at *C
116.8. Some of the signals are shown in (Table 1). Based
on above evidence the structure of compound 1 was
identified as 3$-cholest-5-ene-3, 23diol.
Compound 2 was obtained as white crystal, its
melting point is 106-108ºC, Rf value is 0.350
(Hexane/Ethylacetate). The structure was established by
DEPT, 1H-NMR and 13C-NMR at 400MHz in CDCl3. Its
HO Compound 2
RESULTS AND DISCUSSION
Thin layer Chromatography of Compound 1 and
Compound 2 (Fig. 1). Chromatographic separation of the
acetone fraction over a silica gel G (60-120) mesh size led
to isolation of two compounds that gave positiv
Salkowski and Liebermann-Burchard test specific for
steroids.
Compound 1 was obtained as white crystal, its
melting point is 228-230ºC, Rf value is 0.515
(Hexane/Ethylacetate). The structure was established by
1
H-NMR and 13C-NMR at 600MHz in CDCl3. The 1H-N
spectrum of the isolated compound showed a series of
271
Br. J. Pharmacol. Toxicol., 2(5): 270-272, 2011
HNMR revealed the presence of hydroxyl group at *H
3.52 while the oleifinic proton appear at *H 5.35 which
shows that there is double bond between C-5 and C-6.
The spectra further revealed the presence of six methyl
groups *H 0.69. 1.03, 0.92, 0.82, 0.82, and 0.85
corresponding to *C C-18, C-19, C-21 C-26, C-27, and C29, repectively. The 13C NMR spectrum showed the
presence of 29 carbon signals in the molecules. The
DEPT spectrum exhibited six methyl, eleven methylene
and nine methine, while the remaining three signals in the
broad band spectrum were due to the quaternary carbon
atom. All the signals are shown in (Table 1). Base on the
evidence above compound 2 was identified as 24-ethyl
cholest-5ene-3$-ol. These two compounds probably could
be responsible for the analgesic and anti-inflammatory
activity already reported.
1
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