New steroidal glycosides from the stem bark of Mimusops elengi

Article abstract of DOI:10.1007/s10600-010-9673-7

Two new steroidal glycosides (1 and 2) have been isolated from the ethanolic extract of the stem bark of Mimusops elengi L. and characterized as stigmasta-5,22-dien-3β-ol-3β-D-glucuropyranosyl-(6'β→1'') -Dglucopyranoside (1) and β-sitosterol-3β-(3''',6'''

Full text of DOI:10.1007/s10600-010-9673-7

Chemistry of Natural Compounds, Vol. 46, No. 4, 2010
NEW STEROIDAL GLYCOSIDES FROM THE STEM
BARK OF Mimusops elengi
1
2*
1
N. Akhtar, M. Ali, and M. S. Alam
UDC 547.918
Two new steroidal glycosides (1 and 2) have been isolated from the ethanolic extract of the stem bark of
Mimusops elengi L. and characterized as stigmasta-5,22-dien-3ꢀ-ol-3ꢀ-D-glucuropyranosyl-(6ꢁꢀꢂ1ꢁꢁ)-D-
glucopyranoside (1) and ꢀ-sitosterol-3ꢀ-(3ꢁꢁꢁ,6ꢁꢁꢁ,7ꢁꢁꢁ-trihydroxynaphthyl-2ꢁꢁꢁ-carboxyl)-4ꢁꢁ-
glucopyranosyl(1ꢁꢁꢂ4ꢁ)-glucopyranoside (2) along with the known compounds stigmasta-5-en-3ꢀ-ol,
lup-20(29)-en-3ꢀ-ol, and stigmasta-5-en-3ꢀ-D-glucopyranoside. Their structures have been elucidated on
the basis of spectral data analysis and chemical reactions.
Keywords: Mimusops elengi L., Sapotaceae, stem bark, steroidal glycosides.
Mimusops elengi L. (Sapotaceae) is an ornamental small tree with sweet scented flowers growing wild throughout the
greater part of India [1]. All parts of the plant are prescribed in the indigenous system of medicine as a febrifuge, astringent,
purgative, and stimulant [2]. The decoction of the bark is used as a gargle [3]. It exhibited antimicrobial, antiulcer, hypotensive,
anti-HIV, and spasmolytic activities [4–8]. Ursolic acid, betulinic acid, the fatty acid ester of ꢃ-spinasterol [9], 3ꢀ,6ꢀ,19ꢃ,23-
tetrahydroxyurs-12-ene, 1ꢀ-hydroxy-3ꢀ-hexanoyllup-20(29)-ene-23,28-dioic acid, and bassic acid have been isolated from
the bark [10, 11]. This paper deals with the isolation and structural elucidation of two new steroidal glycosides from the stem
bark of Mimusops elengi L.
Compound 1, designated as stigmasterol-ꢀ-D-glycopyranoside, was obtained as a colorless amorphous powder from
chloroform–methanol (19:1) eluants. It gave a positive test for sterol glycosides. Its IR spectrum exhibited absorption bands
–1
–1
–1
for hydroxyl groups (3450, 3380, 3260 cm ), ester group (1738 cm ), and unsaturation (1614 cm ). On the basis of mass
13
and C NMR spectra, its molecular weight was established at m/z 750, consistent with the molecular formula of a sterol
diglucoside, C H O , and confirmed by HR-MS 751.9713 [M + H] . Its mass spectrum showed important ion fragments
appearing at m/z 411 [M – C H O , diglycoside] , 271 [411 – C H , side chain] , 255 [271 – Me] , and 239 [255 – Me] .
+
41 66 12
+
+
+
+
12 19 11
10 19
These fragments suggested that it was a C sterol possessing one double bond in the carbocyclic ring system and one double
29
bond in the side chain. The mass spectrum indicated the presence of an ethyl group in the side chain, which was placed at
C-24 on the basis of biosynthesis analogy as well as similarities in chemical shifts of protons and carbons of the side chain with
+
+
the related compounds. The ion fragments at m/z 339 [M – 411] and 179 [C H O ] suggested the glucuronosyl glucoside
6
11 6
1
nature of the carbohydrate moiety. The H NMR spectrum of 1 exhibited two one-proton multiplets at ꢄ 5.07 and 5.28 assigned
to vinylic H-22 and H-23. A one-proton broad signal at ꢄ 5.30 was ascribed to vinylic H-6. A one-proton broad multiplet at ꢄ
3.85 with half-width 16.5 Hz was attributed to ꢃ-oriented C-3 carbinol proton. Two one-proton doublets at ꢄ 4.95 (J = 7.2 Hz)
and 4.88 (J = 7.0 Hz) were attributed to H-1ꢁ and H-1ꢁꢁ anomeric protons, respectively. A two-proton broad signal at ꢄ 3.33 was
associated with oxygenated C-6ꢁꢁ methylene protons. The remaining hydroxymethine protons of the glycosidic linkage appeared
between ꢄ 4.50–3.35. Two three-proton broad signals at ꢄ 0.66 and 1.19 were assigned to C-18 and C-19 tertiary methyl
protons. Four doublets at ꢄ 0.96 (J = 6.1 Hz), 0.86 (J = 8.4 Hz), 0.81 (J = 6.3 Hz), and 0.83 (J = 6.3 Hz), all integrating for three
protons each, were due to C-21, C-26, and C-27 secondary and C-29 primary methyl protons, respectively. The presence of all
the methyl signals in the range ꢄ 1.19–0.66 suggested the location of these functionalities on the saturated carbons.
1) Department of Chemistry, Faculty of Science; 2) Department of Pharmacognosy and Phytochemistry, Faculty
of Pharmacy, Jamia Hamdard (Hamdard University) New Delhi-110 062, India, e-mail: mali_hamdard@yahoo.co.in. Published
in Khimiya Prirodnykh Soedinenii, No. 4, pp. 465–468, July–August, 2010. Original article submitted March 3, 2009.
0009-3130/10/4604-0549 02010 Springer Science+Business Media, Inc.
549

1
13
TABLE 1. H (400 MHz) and C (100 MHz) NMR Data for Compounds 1 and 2
ꢄH (J/Hz)
ꢄC
C atom
1
2
1
2
1
2
3
4
5
6
7
8
1.34 m, 2.94 m
1.86 m, 1.82 m
3.85 br.m (w1/2 = 16.5)
1.34 m, 2.26 m
1.79 m, 1.73 m
3.09 br.m (w1/2 = 16.5)
37.43
31.75
74.94
43.56
138.58
119.42
32.17
33.86
53.82
35.28
22.81
38.53
39.41
54.58
24.98
29.71
59.17
12.17
20.46
34.52
18.93
125.96
127.79
48.01
25.41
20.93
22.17
23.85
9.89
110.04
87.13
68.84
71.62
85.45
170.85
102.48
76.51
68.84
71.69
80.43
61.98
–
38.95
27.68
73.36
40.03
141.23
121.04
31.28
33.58
49.49
36.72
39.76
38.28
41.50
56.05
25.42
28.99
55.33
11.43
19.56
36.09
18.44
33.25
35.42
45.08
29.06
18.93
20.47
23.73
11.48
107.17
78.32
70.00
76.68
79.76
61.08
100.79
77.02
73.00
69.98
78.76
60.72
112.21
140.22
158.44
111.35
110.35
151.97
148.10
110.35
139.58
135.94
173.16
2.24 m, 1.99 m
2.87 m, 2.22 br.s
–
–
5.30 br.s
2.17 m, 1.99 m
5.17 m
1.67 m, 2.41 m
1.24 m
1.54 m
1.16 m
1.51 m
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1ꢁ
–
–
2.17 m, 1.52 m
1.07 m, 1.82 m
2.02 m, 1.49 m
1.01 m, 1.89 m
–
–
1.15 m
1.22 br.s, 1.55 br.s
1.63 m, 1.50 m
1.15 m
1.03 m, 1.63 m
1.67 m, 1.34 m
1.39 m
0.66 br.s
1.19 br.s
2.53 m
1.39 m
0.60 br.s
1.11 br.s
2.02 m
0.96 d (J =6.1)
0.81d (J = 6.5)
1.63 m, 1.06 m
1.29 m, 1.79 m
1.29 m
1.63 m
0.77 d (J = 6.0)
0.71 d (J = 6.3)
1.15 m, 1.67 m
0.73 d (J = 5.7)
4.12 d (J = 7.5)
5.07 m
5.28 m
2.27 m, 1.99 m
1.54 m
0.86 d (J = 8.4)
0.81 d (J = 6.3)
1.26 m, 1.63 m
0.83 d (J = 6.3)
4.95 d (J = 7.2)
4.08 m
3.35m
4.06 m
4.50 m
3.38 m
3.41 m
3.85 m
3.94 br.s
2ꢁ
3ꢁ
ꢁ
4
5ꢁ
6ꢁ
–
3.07 d (J = 8.22), 3.04 d (J = 8.22)
4.88 d (J = 7.0)
4.14 d (J = 7.5)
ꢁꢁ
1
4.08 m
3.35 m
4.06 m
4.26 m
3.38 m
3.41 m
3.38 m
3.94 br.s
2ꢁꢁ
3ꢁꢁ
4ꢁꢁ
ꢁꢁ
5
3.33 br.s, 3.33 br.s
3.07 d (J = 8.22), 3.04 d (J = 8.22)
6ꢁꢁ
1ꢁꢁꢁ
2ꢁꢁꢁ
–
–
–
–
–
–
–
–
–
–
–
7.32 br.s
–
–
–
–
–
–
–
–
–
–
ꢁꢁꢁ
3
7.32 br.s
7.38 br.s
4ꢁꢁꢁ
5ꢁꢁꢁ
6ꢁꢁꢁ
–
–
ꢁꢁꢁ
7
8ꢁꢁꢁ
9ꢁꢁꢁ
10ꢁꢁꢁ
7.38 br.s
–
–
–
–
–
ꢁꢁꢁ
11
______
Coupling constants in Hz are given in parentheses.
550

26
27
29
21
18
21
18
22
20
17
17
29
26
19
5
19
14
1
4
14
1
3
3
6'
HO
O
HO
CO
OH
O
O
O
6
6
O
O
O
1'
OH
OH
6''
HO
O
HO
HO
OH
H
O
OH
OH
1''
OH
OH
1'''
HO
CO
7'''
2'''
2
1
HO
3''' OH
6'''
13
The C NMR spectrum of 1 showed important signals for the ester carbon at ꢄ 170.85 (C-6ꢁ), the vinylic carbon at ꢄ 138.58
(C-5), 119.42 (C-6), 125.96 (C-22), and 127.79 (C-23), the carbinol carbon at ꢄ 74.94 (C-3), anomeric carbons at 110.04 (C-1ꢁ)
and 102.48 (C-1ꢁꢁ), sugar carbons between ꢄ 85.54–61.98, and the remaining methyl, methylene, and methine carbons between ꢄ
59.17–9.89. The existence of the ester signal at ꢄ 170.85 suggested that the anomeric proton of the glucose moiety was
1
13
esterified with the carboxylic group of the glucuronic acid forming the (1ꢁꢁꢀꢂ6ꢁ) linkage. The H and C NMR values of the
steroidal nucleus of 1 were compared with the related stigmasterols [12, 13]. The HMBC spectrum of 1 showed correlations of
C-3 with H -2, H -4, and H-1ꢁ; C-6 with H -7; and C-22 with H-20, H -21, and H-23. Acid hydrolysis of 1 yielded stigmasterol,
2
2
2
3
D-glucose, and D-glucuronic acid (TLC comparable). On the basis of these findings, the structure of 1 has been established as
stigmasta-5,22-dien-3ꢀ-ol-3ꢀ-D-glucuropyranosyl-(6ꢁꢀꢂ1ꢁꢁ)-D-glucopyranoside. This is a new steroidal glycoside isolated
for the first time from a natural source.
Compound 2, named ꢀ-sitosterol diglucosyl naphthoate, was obtained as a colorless amorphous powder from
chloroform–methanol (93:7) eluats. It responded positively to the tests for sterol glycosides. Its IR spectrum exhibited absorption
–1
–1
–1
bands for hydroxyl groups (3540, 3400, 3365 cm ), ester group (1741cm ), and unsaturation (1610 cm ). On the basis of
13
mass and C NMR spectra, its molecular weight was established at 940, consistent with the molecular formula C H O for
52 76 15
a carboxynaphthalene substituted diglucoside of ꢀ-sitosterol, and confirmed by HR-MS at m/z 941.1714. Its mass spectrum
+
+
showed ion fragments generated at m/z 413 [M – glycoside] , 527 [C H O ; Glu ꢂ Glu ꢂ C H O ] , 575 [ꢀ-sitosterol ꢂ
23 27 14
11 7 4
+
+
glucoside] , and 365 [C H O ; Glu ꢂ C H O ] , indicating that a diglucoside linked with a trihydroxynaphthalenic
17 17
9
11 7 4
1
carboxylate was attached to ꢀ-sitosterol. The H NMR spectrum of 2 showed two broad signals at ꢄ 7.32 and 7.38, both
integrating for two protons each, assigned to H-1ꢁꢁꢁ and H-4ꢁꢁꢁ and H-5ꢁꢁꢁ and H-8ꢁꢁꢁ aromatic protons, respectively. A one-
proton multiplet at ꢄ 5.17 was ascribed to vinylic proton H-6. Two one- proton doublets at ꢄ 4.12 (J = 7.5 Hz) and 4.14 (J = 7.5
Hz) were attributed to H-1ꢁ and H-1ꢁꢁ anomeric protons, respectively. A one-proton broad multiplet at ꢄ 3.09 with half-width 16.5
Hz was attributed to the ꢃ-oriented H-3 carbinol proton. The hydroxymethine proton of the sugar moiety appeared
between ꢄ 3.94–3.38. Two doublets at ꢄ 3.07 (J = 8.22 Hz) and 3.04 (J = 8.22 Hz), both integrating for two protons each, were
associated with the H -6ꢁ and H -6ꢁꢁ hydroxymethylene proton. Two three-proton broad signals at ꢄ 0.60 and 1.11 were
2
2
assigned to C-18 and C-19 tertiary methyl protons of the sterol moiety. Four doublets at ꢄ 0.81 (J = 6.5 Hz), 0.77 (J = 6.0 Hz),
0.71 (J = 6.3 Hz), and 0.73 (J = 5.71 Hz), all integrating for three protons each, were attributed to C-21, C-26, C-27 secondary,
and C-29 primary methyl protons, respectively. The remaining methylene and methine protons resonated as multiplets between
ꢄ 2.41–1.06. The existence of all the methyl protons in the range ꢄ 1.11–0.50 indicated that all these functionalities were
13
attached on the saturated carbons. The C NMR spectrum of 2 exhibited signals for the ester carbon in the deshielded range
at ꢄ 173.16, aromatic carbons between ꢄ 158.44–110.35, vinylic carbons at ꢄ 141.23 (C-5) and 121.04 (C-6), and the remaining
1
13
carbons of the ꢀ-sitosterol framework from ꢄ 56.05 to 11.48. The H and C NMR values of the steroidal nucleus were
compared with ꢀ-sitosterol, stigmast-4-en-6ꢀ-ol-3-one [12, 13], and lawsaritol [14]. The HMBC spectrum of 2 exhibited
correlation of C-3 with H -2, H -4 and H-1ꢁ; C-4ꢁ with H-6ꢁ and H-1ꢁꢁ; and C-11ꢁꢁꢁ with H-4ꢁꢁ and H-1ꢁꢁꢁ. Acid hydrolysis of 2
2
2
551

yielded ꢀ-sitosterol and D-glucose (TLC comparable). On the basis of the above-mentioned discussion, the structure of 2 has
been elucidated as ꢀ-sitosterol-3ꢀ-(3ꢁꢁꢁ,6ꢁꢁꢁ,7ꢁꢁꢁ-trihydroxynaphthyl-2ꢁꢁꢁ-carboxyl)-4ꢁꢁ-glucopyranosyl(1ꢁꢁꢂ4ꢁ)-glucopyranoside.
This is a new steroidal glycoside isolated from a natural or synthetic source for the first time. The known compounds have
been identified as stigmast-5en-3ꢀ-ol (ꢀ-sitosterol) [15, 16], lup-20(29)-en-3ꢀ-ol(lupeol) [16, 17], and stigmast-5en-3ꢀ-ol-ꢀ-
D-glucopyranoside (ꢀ-sitosterol glucoside) [16, 18].
EXPERIMENTAL
General Methods. Melting points were determined on a Perfit melting point apparatus (Ambala, India) and are
uncorrected. IR spectra were recorded on KBr discs, using a Bio-Rad FT-IR 5000 spectrometer (FTS 135, Hongkong).
1
13
UV spectra were measured with a Lambda Bio 20 spectrophotometer (Perkin–Elmer, Switzerland) in methanol. H and C NMR
spectra were scanned using Bruker Advance DRY 400 spectrospin and Bruker Advance DRY 100 spectrospin instruments
(Germany), respectively, in CDCl and TMS as an internal standard. FAB-MS spectra were obtained using a JEOL-JMS-DX
3
303 spectrometer (USA). Column chromatography was performed on silica gel (Qualigens, Mumbai, India) 60–120 mesh.
TLC was run on silica gel G (Qualigens, Mumbai, India). Spots were visualized by exposure to iodine vapor, UV radiation,
and by spraying reagents.
Plant Material. The barks of Mimusops elengi were purchased from the Khari Baoli local market of Delhi and
authenticated by Dr. H. B. Singh, taxonomist, NISCAIR, CSIR, New Delhi. A voucher specimen, No. PRL/JH/03/05, was
deposited in the Herbarium of the Phytochemical Research Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi.
Extraction and Isolation. The bark of Mimusops elengi was dried in an oven at 45ꢅC for 2–3 days and coarsely
powdered. The ground bark (3 kg) was extracted with ethanol in a Soxhlet apparatus. The ethanol extract was concentrated
under reduced pressure to yield a dark brown viscous mass (120 g, 4.0%). The extract was dissolved in a minimum amount of
methanol and adsorbed on silica gel (60–120 mesh) for preparation of slurry. The air-dried slurry was chromatographed over
the silica gel column packed in petroleum ether (60–80ꢅC). The column was eluted with petroleum ether, petroleum ether–
chloroform (9:1, 3:1, 1:1, 1:3 v/v), chloroform, chloroform–methanol (99:1, 98:2, 95:5, 9:1, 3:1, 1:1, 1:3 v/v), and methanol,
successively, in order of increasing polarity to isolate the following compounds.
Compound 1. Elution of the column with chloroform–methanol (99:1) yielded a colorless amorphous powder of 1,
recrystallized from methanol; 410 mg (0.013%); R 0.68 (toluene–ethyl formate–formic acid 10:10:3); mp 255–256ꢅC. UV
f
–1
(MeOH,
, nm): 247 (log ꢆ 5.7). IR (KBr, ꢇ , cm ): 3450, 3380, 3260, 2920, 2846, 1738, 1614, 1578, 1465, 1354, 1168,
max
1
max
13
+
1106, 989. H NMR, see Table 1. C NMR, see Table 1. +ve FAB-MS m/z (rel. int.): 750 [M] (C H O ) (1.1), 411 (15.3),
41 66 12
397 (55.3), 395 (35.0), 379 (10.1), 353 (8.2), 339 (7.9), 271 (11.2), 255 (18.6), 239 (19.2), 215 (20.6), 189 (27.1), 179 (30.8),
+
161 (46.2), 145 (49.8), 121 (69.1), 95 (100). HR-MS: 751.9713 [M + H] (calcd for C H O 751.9702).
41 67 12
Acid Hydrolysis of 1. Compound 1 (20 mg) was dissolved in MeOH and 2N HCl (1:1) and the solution heated up to
half volume left. The solution was extracted with EtOAc (3 ꢈ 10 mL), washed with water (2 ꢈ 10 mL), dried over Na SO , and
2
4
evaporated to obtain the stigmasterol, mp 168–169ꢅC, co-TLC comparable. The aqueous phase was concentrated and subjected
to paper chromatography using n-butanol–ethanol–water (4:1:2.2) as the developing solvent system for standard samples of
monosaccharide. The paper was sprayed with aniline hydrogen phthalate and the sugars were identified as D-glucose and
D-glucuronic acid.
Compound 2. Elution of the column with chloroform–methanol (93:7) produced a pale yellow amorphous powder of
2, recrystallized from methanol; 100 mg (0.003%); R 0.50 (toluene–ethyl acetate–formic acid 5:4:1); mp 279–280ꢅC (decomp.).
f
–1
UV (MeOH,
, nm): 253, 364 (log ꢆ 5.7, 1.1). IR (KBr, ꢇ , cm ): 3540, 3400, 3365, 2943, 2846, 1741, 1610, 1490,
max
max
1
13
+
1420, 1367, 1097, 1063, 758. H NMR, see Table 1. C NMR, see Table 1. +ve FAB-MS m/z (rel. int.): 940 [M] (C H O ),
52 76 15
575 (5.2), 527 (13.2), 413 (11.6), 397 (100), 381 (12.3), 365 (16.3), 273 (9.8), 255 (9.6), 240 (8.3), 136 (55.3), 119 (12.6), 107
+
(21.3). HR-MS: 941.1712 [M + H] (calcd for C H O 941.1723).
52 76 15
Alkaline Hydrolysis of 2. Compound 2 (20 mg) was dissolved in MeOH and 1 N KOH (1:1) and heated for 1 hour.
The solution was extracted with EtOAc (3 ꢈ 10 mL), and washed with water (2 ꢈ 10 mL), dried over Na SO , and evaporated
2
4
to give ꢀ-sitosterol, mp 138–139ꢅC, co-TLC comparable. The aqueous phase was neutralized with dil. HCl, concentrated, and
analyzed by paper chromatography along with standard samples of monosaccharides. n-Butanol–ethanol–water (4:1:2.2) was
used as the developing solvent system. The paper was sprayed with aniline hydrogen phthalate. The sugar was identified as
D-glucose.
552

ACKNOWLEDGMENT
The authors are thankful to the Head, Regional Sophisticated Center, Central Drug Research Institute, Lucknow for
recording the spectral data of the compounds.
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