Isolation and characterization of β-sitosterol-D-glycoside from petroleum extract of the leaves of Ocimum sanctum L

Abstract

β-sitosterol-D-glycoside was isolated from the petroleum ether extract of the leaves of Ocimum sanctum L. This compound has not been previously isolated or reported from the leaves of this variety. The structures of β-sitosterol-D-glycoside are elucidated with the help of UV, IR, 1H-NMR, 13C-NMR, spectral data.

Full text

As. J. Food Ag-Ind. 2009, 2(01), 39-43
Asian Journal of
Food and Agro-Industry
ISSN 1906-3040
Available online at www.ajofai.info
Short communication
Isolation and characterization of β-sitosterol-D-glycoside from
petroleum extract of the leaves of Ocimum sanctum L
S. M. Mizanur Rahmana*, Zinnat Arzu Muktaa and M. Amzad Hossainb
aDepartment of Chemistry, University of Dhaka Dhaka-1000, Bangladesh
bChemistry Division, Atomic Energy Centre, Ramna, Dhaka-1000, Bangladesh.
*Author to whom correspondence should be addressed, email: smmrdu@yahoo.com
Abstract. β-sitosterol-D-glycoside was isolated from the petroleum ether extract of the leaves
of Ocimum sanctum L. This compound has not been previously isolated or reported from the
leaves of this variety. The structures of β-sitosterol-D-glycoside are elucidated with the help
of UV, IR, 1H-NMR, 13C-NMR, spectral data.
Keywords: biochemistry, bioactive compounds, isolation, basil, Bangladesh, spectral
analysis.
Introduction
Ocimum sanctum L. plant is a shrub reaching a height of 0.5 to 1.5m. The plant is locally
known as Tulshi throughout the Indo-Bangla subcontinent, while the English name is Holy
Basil. The leaves are 2-4cm in length. Among the several varieties of the plant, one of the
most commonly used has dark leaves. The inflorescence is a long spike with tiny purple
flowers and the plant has a stronger smell. Different parts of the plant have been traditionally
used for the treatment of various diseases such as reducing the blood glucose level as well as
reduction of total cholesterol in the blood [1]. Its antioxidant properties have also been
reported [2]. It has also been reported [3, 4] that the leaves and seeds of this variety of
Ocimum basilicum L. contain essential oils [5, 10] and the volatile oil of the seed is
composed of fatty acids and β-sitosterol, while in addition the seed mucilage has some level
of sugars [5, 10].
In the present investigation, we describe the isolation of β-sitosterol-D-glycoside from the
petroleum ether extract of the leaves of Ocimum sanctum L. To the best of our knowledge
this compound has not been previously isolated or reported from the leaves of this variety.

As. J. Food Ag-Ind. 2009, 2(01) 40
Materials and Methods
Plant material
The plant materials (including the root) of Ocimum sanctum L. were collected from the
nursery of the Bangladesh Agricultural Development Corporation at Gazipur. The plant was
identified from the Department of Botany of Dhaka University. The collected fresh plant
materials were cleaned thoroughly and after separation, the leaves from the stem were
initially dried under sunlight, followed by controlled drying in an electrical oven at 40oC. The
dried plant materials were chopped into small pieces followed by grinding through a Cyclotec
grinder (200 mesh). These fine powdered materials were used in the present investigation.
Spectroscopic investigation
Melting point was determined by an electrochemical micro-melting point apparatus
(Gallenkamp). The UV, IR (KBr) spectra were recorded on a Shimadzu UV-168A and
Shimadzu IR-470A spectrophotometer, respectively. The 1H-NMR and 13C-NMR spectra
were recorded on a Bruker R-32 (400 MHz) in deuterated methanol (CD3OD) with TMS as
an internal standard (chemical shifts in δ, ppm). TLC was performed with silica gel GF254.
All solvents were analytical reagent grade.
Extraction and isolation
The leaf powder (80g) was exhaustively extracted in a soxhlet apparatus with methanol.
After filtration the crude extract was concentrated under reduced pressure at 40oC in a rotary
evaporator. After the removal of methanol the dried mass was suspended in water and further
extracted successively with hexane, chloroform, ethyl acetate and n-butanol, respectively. All
these extracts were collected separately and preserved for analysis. The hexane soluble
fraction was concentrated to dry mass under reduced pressure at 40oC in a rotary evaporator.
The concentrated extract was dried by vacuum pump to yield dry mass (18.0g). The dry mass
was mixed with a small amount of silica gel (60-120 mesh) maintaining the ratio (2:1) and
dried in air. After drying the mixture was powdered in a mortar and applied to vacuum liquid
chromatography (VLC) over TLC grade silica gel (GF254). The column was initially eluted
with petroleum ether (40-60oC) followed by gradient elution with the mixture of petroleum
ether with an increasing amount of dichloromethane. These elutes were collected in a series
of test tubes (more than 170 tubes) with 20ml in each fraction. All of these fractions were
monitored by TLC (over silica gel GF254). The elutes of similar behaviour (similar Rf values)
were combined together to afford seven fractions F1 (1-12), F2 (17-19), F3 (32-35), F4 (62-
64), F5 (92-100), F6 (101-103), F7 (119-121). All of these fractions were concentrated
separately and allowed to stand at room temperature for a few weeks. A yellowish semi-solid
amorphous substance (10.0 mg) settled out from fraction F4 and this fraction was marked as
SP1.
Characterization of the compound SP1
A yellowish semi-solid amorphous substance (10.0 mg) was obtained from the fraction F4. It
could not be crystallized from any solvent. It was soluble in petroleum ether, ethyl acetate,
chloroform and dichloromethane. It was further purified by preparative TLC over silica gel
GF254 using petroleum ether-dichloromethane (2:3) as a developing solvent. Rf was found to
be 0.60 and the compound was visualized as a yellow coloured single spot upon its exposure
to iodine chamber and as a violet colour on spraying with vanillin-sulphuric acid reagent
followed by heating in an electric oven at 110oC. The structures according to IR were found
to be as follows:

As. J. Food Ag-Ind. 2009, 2(01) 41
(3600-3400), 2900, 2850, 1720, 1640, 1450, 1240, 900, (830-800) cm-1; 1H-NMR (400 Mz,
CDCl3+ CD3OD): 7.25 (s, 1-H, –OH), 6.67-6.84 (m,1H proton of sugar moiety), 5.47 (s, 1H,
H-6), 5.35 (dd, 1H, J=12.5 and 8.5 Hz, H-23), 5.03-5.08 (dd, 1H, J= 12.5 and 8.5 Hz, H-22),
4.96 ( s, 1H, proton of sugar moiety), 4.85 (s, 1H, anomeric proton), 3.86 (m, 1H, H-3) 2.03-
3.31 (m 3H, proton of sugar moiety), 1.24 (s, 3H, H-19), 1.0 (d, 3H, J=6.5 Hz, H-21), 0.97 (t,
3H, J=7.1 Hz, H-29), 8.8 (s, 3H, H-27), 8.7 (s, 3H, H-26), and 0.85 (s, 3H, H-18) ; 13C-NMR
(400 Mz, CDCl3+ CD3OD): 39.9 (C-1), 29.9 (C-2), 77.3 (C-3), 39.8 (C-4), 55.8 (C-5), 21.6
(C-6),39.2 (C-7), 29.7 (C-8), 48.7 (C-9), 29.4 (C-10), 21.6 (C-11), 27.2 (C-12), 50.8 (C-13),
30.2 (C-14), 62.1 (C-15), 77.3 (C-16), 124.3 (C-17), 118.26 (C-18), 130.2 (C-19), 151.87 (C-
20), 178.91 (C-21), 146.47 (C-22), 29.2 (C-23), 14.1 (C-24), 62.12 (C-25), 76.7 (C-26),
63.75 (C-27), 184.9 (C-28), 111.14 (C-29), 121.2(C-30) and the chemical shifts (210.3,
209.45, 130,24, 130.0 and 143.96) ppm are due to the carbon of the sugar moiety.
Results and Discussion
β-sitosterol and some level of sugar have already been reported from the seeds of one of the
varieties of basil [3, 4] but β-sitosteryl-D-glycoside from the leaves of Ocimum sanctum L.
plant was isolated and reported for the first time. Compound SP1 was obtained as a yellowish
amorphous solid. Its IR spectrum showed an absorption peak in the region (3600-33400) cm-1
indicating the presence of a hydroxyl group (-OH) and the absorption bands at 2900-2850 cm-
1 indicated the presence of –CH aliphatic asymmetric stretching of –CH3, –CH2– and > CH2
groups. The absorption band at 1720 cm-1 indicated the presence of (>C=O) stretching of
normal aliphatic ester. The absorption band at 1240 cm-1 indicated the presence of C–N
stretching. The absorption peak at 900 cm-1 indicated the aromatic stretching (out of plane
bending). Finally, the absorption band at 830 and 800 cm-1 indicated the –CH stretching of
>C=C-H group. The 1H-NMR spectrum showed the chemical shift at δ 0.85 and 1.24
indicated the presence of two angular methyl signals. The proton NMR spectrum also
exhibited one olefinic double bond proton as a doublet at δ 5.35, along with the two up field
signals at δ 0.87 and 0.88 respectively, due to the presence of two secondary methyl groups at
position 26 and 27 of the skeleton, i.e, the presence of an isopropenyl group of the molecular
structure. The very up field chemical shift at δ 0.97 as a triplet with the intensity of 3H and
coupling constant of J=7.1 Hz was assigned for the terminal methyl group of 29. Similarly,
the other up field chemical shift at δ 1.0 with the coupling constant J=6.5 Hz of 3H intensity
was assigned the secondary methyl group at position 21 of the molecular structure. The
chemical shifts in the region δ 2.03-3.31 as a multiplet was assigned the presence of five
protons of the sugar moiety and the very downfield chemical shift at δ 7.25 was assigned for
the proton of OH group of glycoside. The 13C-NMR spectra of the compound SP1 revealed
the presence of 29 carbons, the chemical shift at δ 76.7 and 63.8 were assigned for the two
separate terminal methyl groups linked at position 25 of the molecular structure. The three
downfield chemical shifts at δ 128.3, 130.2 and 178.9 respectively, were assigned for the
angular methyl carbons linked at C-18, C-19 and C-21 position. The up field signals at δ
29.7, 29.4 and 30.4 were assignable to the carbon at positions 8, 10 and 14 that was fused in
the proposed β-sitosteryl-D-glycoside derivative. Similarly, the relative down field chemical
shifts at δ 48.7, 50.8 and 55.8, respectively, were assigned for the carbon that was fused at
positions C-9, C-13 and C-5, respectively, in the proposed skeleton. The up field chemical
shift at δ 39.9, 29.9 77.3, 39.8,21.6, 39.2, 21.6, 27.2, 62.1 and 77.3 were appropriate for the
cyclohexyl and cyclopentyl carbons at positions 1, 2, 3, 4, 6, 7, 11, 12, 15 and 16,
respectively. The other shifts at δ 151.87, 146.47, 29.2, 14.1, 62 12 and 184.9 were assigned
for the carbon numbers 20, 22, 23, 24, 25 and 28, respectively, which constitute the side

As. J. Food Ag-Ind. 2009, 2(01) 42
chain of six carbons which were linked at position 17 of the cyclopentyl ring. The chemical
shift at δ 124.3 was assigned for the carbon number 17 which was the point of link of a side
chain to the cyclopentyl ring. The very down field chemical shift at 210.3, 209.45, 130.24,
130.0 and 143.96 were assigned for the carbon of the sugar moiety.
On the basis of IR, 1H-NMR, 13C-NMR, spectral data and the other physical properties the
isolated pure compound SP1 were identified and established as β-sitosterol-D-glycoside as
shown in Fig. 1.
1
2
3
4
5
10
19
11
12
9
8
6
7
13
14
15
16
17
18 20
21 22
23
24
25
27
26
28
29
Glyco
β-Sitosteryl-D-glycosideFigure: 1
Acknowledgements
The authors are grateful to Ms. Aminul Ahsan, Senior Scientific Officer, Analytical
Chemistry Division, BCSIR Laboratories, Bangladesh for her help in connection with 1H-
NMR, 13C-NMR, They are also grateful to Dr. S. M. Salehuddin, Chemistry Division,
Bangladesh Atomic Energy Centre, for his help in connection with GC-MS/MS.
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