New triterpenoids from Morus alba L. stem bark

Article abstract of DOI:10.1080/14786419.2012.676547

Two lupeol-type pentacyclic triterpenoids characterised as lup-20(29)-en-3β-ol-27-oic acid (moruslupenoic acid A) and lup-12, 20(29)-dien-3β-ol-26-oic acid (moruslupenoic acid B) and lanst-5, 24-dien-3β-yl acetate (moruslanosteryl acetate) along with the known triterpenoidal phytoconstituents α-amyrin acetate, β-amyrin-β-D- glucopyranoside and betulinic acid have been isolated from the stem bark of Morus alba L. (Moraceae). The structures of the isolated phytoconstituents were established on the basis of spectral data analysis and chemical means.

Full text of DOI:10.1080/14786419.2012.676547

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New triterpenoids from Morus alba L.
stem bark
Abuzer Ali a & Mohd. Ali a
a Department of Pharmacognosy and Phytochemistry, Faculty of
Pharmacy, Jamia Hamdard, New Delhi 110062, India
Version of record first published: 05 Apr 2012.
To cite this article: Abuzer Ali & Mohd. Ali (2013): New triterpenoids from Morus alba L. stem bark,
Natural Product Research: Formerly Natural Product Letters, 27:6, 524-531
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Natural Product Research, 2013
Vol. 27, No. 6, 524–531, http://dx.doi.org/10.1080/14786419.2012.676547
New triterpenoids from Morus alba L. stem bark
*
Abuzer Ali and Mohd. Ali
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard,
New Delhi 110062, India
(Received 25 July 2011; final version received 11 February 2012)
Two lupeol-type pentacyclic triterpenoids characterised as lup-20(29)-en-3�-ol-
27-oic acid (moruslupenoic acid A) and lup-12, 20(29)-dien-3�-ol-26-oic acid
(moruslupenoic acid B) and lanst-5, 24-dien-3�-yl acetate (moruslanosteryl
acetate) along with the known triterpenoidal phytoconstituents �-amyrin acetate,
�-amyrin-�-D-glucopyranoside and betulinic acid have been isolated from the
stem bark of Morus alba L. (Moraceae). The structures of the isolated
phytoconstituents were established on the basis of spectral data analysis and
chemical means.
Keywords: Morus alba; Moraceae; stem bark; triterpenoids; moruslupenoic acid
A; moruslupenoic acid B; moruslanosteryl acetate
1. Introduction
Morus alba L. (Moraceae) is a monoecious or dioecious shrub or moderate-sized tree
commonly known as mulberry. The genus Morus consists of over 150 species; among
them, M. alba is dominant (Anonymous, 2001; Butt, Nazir, Sultan, & Schroen, 2008).
Mulberry is found from temperate to subtropical regions of the Northern hemisphere to
the tropics of the Southern hemisphere and grows in a wide range of climatic,
topographical and soil conditions from sea level to altitudes as high as 4000 m. The
plant is indigenous to China and commonly cultivated in Baluchistan, Afghanistan,
northern part of the trans-Indus territory and N.W. Himalaya. The bark of the large stem
is brown, rough, fissures mostly vertical, considered as vermifuge and purgative. Mulberry
is used for its foliage to feed the silkworm (Anonymous, 2001; Ercisli & Orhan, 2007;
Mhaskar, Blatter, & Caius, 2000). Mulberry is a promising nature’s functional tonic and
has a unique nutritional profile containing proteins, phenolics, flavonoids and anthocy-
anins, which enhance its significance. Phytol, �-sitosterol, lanost-7-en-3-on, �-amyrin,
�-amyrin, lupeol, mulberrin, cyclomulberrin, mulberrochomene, cyclomulberrochromene,
mulberranol, albanol A and B have been reported from the stem bark. �-Sitosterol has
inhibitory effect on 5-�-reductase, which has a key role in steroid biosynthesis, whereas
�-amyrin, �-amyrin and lupeol have strong antiinflammatory effect and are a potent
inhibitor of protein kinase in rat liver cells. (Boszormenyi et al., 2009; Butt et al., 2008;
Deshpande, Parthasarathy, & Venkataraman, 1968; Rama Rao, Deshpande, & Shastri,
1983). This article describes the isolation and characterisation of triterpenoids from the
stem bark of M. alba of Delhi region.
*Corresponding author. Email: maliphyto@gmail.com
© 2013 Taylor & Francis

Natural Product Research
525
2. Results and discussion
Six compounds were isolated (Figure 1). Compounds 1, 3 and 6 are the known
triterpenoids identified as �-amyrin acetate (Ali, Ravinder, & Ranachandram, 2000; Sirat,
Susanti, Ahmad, Takayama, & Kitajima, 2010; Wang, Liu, & Xu, 2009), �-amyrin-�-D-
glucopyranoside (Ali, 2001; Mahato & Kundu, 1994) and betulinic acid (Ghosh, Mandal,
A. Chakraborty, Rasul, M.G. Chakraborty, & Saha, 2010; Li et al., 2009; Malik, Ahmad,
Anjum, & Basha, 2010), respectively.
Compound 2, named moruslanosteryl acetate, was obtained as colourless needle
shaped crystals from petroleum ether-chloroform (1:1) eluents. It responded positively to
triterpenoid tests. Its infrared (IR) spectrum exhibited characteristic absorption bands for
ester group at 1730 cm�1 and unsaturation at 1646 cm�1. On the basis of FAB MS and 13
C
NMR spectra, its molecular weight was established at m/z 468 consistent of a molecular
formula of a triterpenic ester, C32H52O2. It had seven degrees of double bond equivalents;
four of them were adjusted in the tetracyclic carbon framework of the triterpene, two in
the vinylic linkage and the remaining one in the ester function. The mass spectrum of 2
exhibited the prominent ion peaks generated at m/z 453 [M�Me]þ, 410 [453�CH3CO]þ,
408 [M�CH3COOH]þ, 393 [408�Me]þ and 378 [393�Me]þ indicating the presence of the
acetate group in the molecule. The ion peaks arising at m/z 111 [C8H15, side chain]þ 357
[M–side chain]þ, 315 [357�ring C]þ, 342 [357�Me]þ, 297 [357�CH3COOH]þ, 282
[297�Me]þ and 267 [282�Me]þ supported the existence of one of the vinylic linkage in the
side chain and another one in the triterpenic nucleus and the acetate group. The ion
fragments produced due to fragmentation of the mass unit 297 at m/z 174 [C8,14–C9,11
fission, C13H18]þ, 188 [C8,14–C11,12 fission, C14H20]þ and 202 [C8,14–C12,13 fission,
C15H22]þ suggested saturated nature of the ring C. The ion peak arising due to
fragmentation of the rings A and B at m/z 142 [C8H14O2]þ, 194 [C12H18O2]þ, 134
[194�CH3COOH]þ, 119 [134�Me]þ, 208 [C7,8–C9,10 fission; C13H20O2]þ, 148
[208�CH3COOH]þ and 133 [148�Me]þ indicated the existence of one of the vinylic
linkage at C-5 and the hydroxyl group in the ring A, which was placed at C-3 by
1
considering biogenetic pathway. The H NMR spectrum of 2 displayed two one-proton
multiplets at � 5.22 and 5.07 assigned to vinylic protons H-6 and H-24, respectively. A one-
proton doublet at � 4.51 with coupling interaction of 5.5 and 9.5 Hz was ascribed
�-oriented H-3 carbinol proton. Three broad signals at � 2.01, 1.65 and 1.57, all integrating
for three protons each, were attributed corresponding to acetyl methyl and to C-26 and
C-27 methyl protons attached to C-25 olefinic carbon. A three-proton doublet at � 0.96
(J ¼ 6.3 Hz) was accounted to C-21 secondary methyl proton. The C-18, C-19, C-28, C-29
and C-30 tertiary methyl protons appeared as three-proton broad signals at � 0.73, 1.10,
0.90, 0.82 and 0.77, respectively. The remaining methine and methylene proton resonated
in the range of � 2.1–1.02. The 13C NMR spectrum of 2 showed important signals
for acetyl carbons at � 170.81 and 21.26, vinylic carbons at � 145.94 (C-5), 125.09 (C-6),
117.55 (C-24) and 130.86 (C-25), oxygenated methine carbon at � 81.08 (C-3) and
1
methyl carbons from � 13.09 to 25.82. The H and 13C NMR spectral data of 2 were
compared with lanosterol-type triterpenoids (Ali, 2001; Ansari, Ali, & Qadary, 1993;
1
Sharma & Ali, 1996). The H-1H COSY spectrum of 2 showed correlation of H-3 with
H2-2, COCH3 and H3-28; H-6 with H2-7 and H-8; and H-24 with H2-23, H3-26 and H3-27.
The HMBC spectrum of 2 exhibited interactions of AcO with H-3; C-5 with H-6, H2-7
and H3-29; and C-25 with H2-23, H-24, H3-26 and H3-27. Alkaline hydrolysis of 2
yielded lup-5, 24-dien-3�-ol. On the basis of the above-mentioned discussion, the structure
of 2 has been formulated as lanst-5, 24-dien-3�-yl acetate. This is a new tetracyclic
triterpenoid.

526
A. Ali and M. Ali
30
20
24
H
22
21
18
29
27
21
22
19
20
17
23
25
26
12
26
12
18
13
14
11
16
15
11
17
13
H
H
25
1
19
28
1
4
9
16
15
14
30
9
2
3
2
3
8
8
10
5
10
27
H
7
5
4
7
CH3COO
CH3COO
6
6
28
29
24
23
α-Amyrin acetate (1)
Moruslanosteryl acetate (2)
30
20
29
21
22
19
29
12
26
18
13
17
20
30
11
27
25
28
16
1
4
21
22
15
19
14
2
9
12
26
18
8
7
10
OH
17
5
3
11
6
13
14
25
1
4
28
O
9
O
16
15
2
3
24
23
1'
8
10
4'
OH
COOH
7
5
27
OH
HO
6
23
OH
24
β-Amyrin-β-D-glucopyranoside (3)
Moruslupenoic acid A (4)
29
29
20
20
30
H
30
11
21
22
21
22
19
19
H
12
18
12
26
17
17
11
9
13
14
26
COOH
H
15
25
25
H
1
4
28
1
COOH
16
28
2
3
16
2
3
8
7
10
8
15
27
H
27
H
5
7
4
OH
HO
6
6
H
23
H
23
24
24
Moruslupenoic acid B (5)
Betulinic acid (6)
Figure 1. Structure of compounds 1–6 isolated from the methanolic extract Morus alba L.
stem bark.
Compound 4, designated as moruslupenoic acid A, was obtained as a colourless
amorphous powder from chloroform-methanol (93 : 7) eluents. It responded positively to
Liebermann-Burchardt test for triterpenoid and yielded effervescences with sodium
bicarbonate solution because of the presence of carboxylic group. Its IR spectrum
exhibited distinctive absorption bands for hydroxyl group (3460 cm�1), carboxylic
function (1686 cm�1) and unsaturation (1642 cm�1). On the basis of FAB MS and 13C
NMR spectra, its molecular weight was established at m/z 456 consistent with the
molecular formula of triterpenic acid, C30H48O3. It indicated seven double bond
equivalents; five of them were adjusted in the pentacyclic skeleton of the pentacyclic
triterpenoid and one each in the vinylic linkage and carboxylic function. The mass
spectrum of 4 showed prominent ion peaks generated at m/z 438 [M�H2O]þ, 397

Natural Product Research
527
[438�C3H5]þ, 411 [M�COOH]þ, 393 [411�H2O]þ, 378 [393�CH3]þ, 441 [M�CH3]þ and
426 [441�CH3]þ supporting the existence of the one each of carboxylic group, hydroxyl
function and isopropenyl group in the molecule. The ion fragments arising at m/z 140
[C5,6–C9,10 fission, C9H16O]þ, 154 [C6,7–C9,10 fission, C10H18O]þ and 168 [C7,8–C9,10
fission, C11H20O]þ indicated saturated nature of the ring A and B and the presence of the
hydroxyl group, which was placed at C-3 on biogenetic anomaly. The ion fragments
formed at m/z 208, 248 [C8,14–C9,11 fission, C14H24O and C16H24O2]þ, 234 [C8,14–C11,12
fission, C15H22O2]þ and 220 [C8,14-C12,13 fission, C14H20O2]þ suggested the existence of
carboxylic function at C27/C28 and saturated nature of the ring C. The ion peaks appearing
at m/z 150, 306 [C14,15–C13,18 fission, C11H18 and C19H30O3]þ, 136 [C13,18–C15,16 fission,
C10H16]þ and 122 [C13,18–C16,17 fission, C9H14]þ indicated the location of carboxylic group
1
at C-27 and isopropenyl chain in ring E at C-19. The H NMR spectrum of 4 displayed
two one-proton broad signals at � 4.68 and 4.56 assigned to unsaturated methylene protons
H2-29. A one-proton double doublet at � 3.50 (J ¼ 5.5, 9.3 Hz) was attributed to �-oriented
carbinol proton H-3. A three-proton broad signal at � 1.65 was accounted C-30 methyl
proton attached at the vinylic carbon C-20. The other methyl signals appeared between �
1.24 and 0.65. The remaining methine and methylene protons resonated in the range of �
2.99–1.17. The 13C NMR spectrum of 4 displayed important signals for carboxylic carbon
at � 177.65 (C-27), vinylic carbons at � 150.67 (C-20) and 109.93 (C-29), carbinol carbon at
1
� 77.38 (C-3) and methyl carbons in the range of � 28.47–14.79. The H and 13C NMR
spectral values were compared with betulinic acid and other related lupene-type terpenoids
(Ali, 2001; Mahato & Kundu, 1994). The DEPT spectrum of 4 showed the presence of 6
methyl, 11 methylene, 6 methine and 7 quaternary carbons. The HMBC spectrum of 4
exhibited correlation of C-3 with H2-2, H2-1 and H3-24; C-27 with H-13 and H2-15; and
C-20 with H2-29, H3-30 and H-19. On the basis of foregoing discussion, the structure of 4
has been elucidated as lup-20(29)-en-3�-ol-27-oic acid. This is a new pentacyclic
triterpenoid.
Compound 5, named moruslupenoic acid B, was obtained as colourless flakes from
chloroform-methanol (93 : 7) eluents. It responded positively to Liebermann-Burchardt
test and produced effervescences with sodium bicarbonate solution. Its IR spectrum
showed characteristic absorption bands for hydroxyl group (3436 cm�1), carboxylic
function (3390, 1686 cm�1) and unsaturation (1638 cm�1). On the basis of FAB MS and
13C NMR spectra, its molecular weight was established at m/z 454 consistent with the
molecular formula of pentacyclic triterpenoic acid, C30H46O3. Its molecular formula
indicated eight double bond equivalents; five of them were adjusted in the pentacyclic
carbon framework of a triterpenoid, two in vinylic linkages and one in the carboxylic
group. The mass spectrum of 5 showed prominent ion peaks generated at m/z 409
[M�COOH]þ, 394 [409�CH3]þ, 379 [304�CH3]þ and 364 [379�CH3]þ supporting the
existence of the carboxylic function in the molecule. The ion peaks arising at m/z 238
[C14H22O3]þ and 216 [C16H24]þ because of retro-Diels-Alder fragmentation indicated the
location of one of the vinylic linkage at C-12 and carboxylic function in ring A/B. The ion
fragments appearing at m/z 220 [238�H2O]þ, 205 [220�CH3]þ, 190 [205�CH3]þ, 193
[238�COOH]þ, 175 [193�H2O]þ, 160 [175�CH3]þ and 145 [160�CH3]þ also supported
the presence of the hydroxyl function in the ring A/B, which was placed at C-3 on the basis
of biogenetic consideration. The ion peaks formed at m/z 175 [216�C3H5]þ, 189
[216�C2H3]þ, 201 [216�CH3]þ, 186 [201�CH3]þ, 159 [186�C2H3]þ and 118 [159�C3H5]þ
1
indicated lupene-type triterpenoid. The H NMR of 5 displayed a one-proton double
doublet at � 5.33 (J ¼ 5.4, 6.0 Hz) assigned to vinylic H-12 proton. Two one-proton broad
signals at � 4.71 and 4.67 were attributed to methylene H2-29. A one-proton double
doublet at � 3.27 with coupling interaction of 5.4 and 10.4 Hz was ascribed to �-oriented

528
A. Ali and M. Ali
carbinol H-3 proton. A three-proton broad signal at � 1.67 was accounted to C-30 methyl
protons attached to C-20 vinylic carbon. Six broad signals between � 1.20 and 0.74, all
located on the saturated carbons were associated with the tertiary methyl protons. The
remaining methylene and methane protons resonated in the range of 2.91–1.15. The 13C
NMR spectrum of 5 showed important signals for carboxylic carbon at � 177.97 (C-26),
vinylic carbons at � 132.51 (C-12), 146.76 (C-13), 150.32 (C-20) and 108.89 (C-29), carbinol
carbon at � 77.98 (C-3), methyl carbons between � 28.62 and 14.21 and the remaining
1
methylene and methine carbons in the range of � 55.61 to 20.42. The H and 13C NMR
spectral data with the lupene type compounds (Ali, 2001; Mahato & Kundu, 1994). The
multiplicity of each carbon was determined by DEPT spectrum of 5. The HMBC spectrum
of 5 exhibited interaction of C-4 with H-3, H2-2, H-5 and H3-24; C-8 with H-9 and H2-7;
C-13 with H-12, H2-11, H3-27 and H-18; and C-20 with H2-29, H3-30 and H-19. On the basis
of spectral data analysis and chemical reactions, the structure of 5 has been established
as lup-12, 20(29)-dien-3�-ol-26-oic acid. This is an unreported lupenic acid. Cyathadonic
acid and epistriatic acid are the lupene-type triterpenoids from Cyathus striatus Willd.
ex, Pers., which possess carboxylic function at C-8 (Ayer, Flanagan, & Reffstrup, 1984).
3. Experimental
3.1. General
Melting points were determined on a Perfit apparatus without correction. The IR spectra
were measured in KBr pellet on a Bio-Red FT-IR spectrometer. Ultraviolet (UV) spectra
were obtained in methanol with a Lambda Bio 20 spectrometer. 1H (300 MHz), 13C
(75 MHz), COSY and HMBC NMR spectra were recorded on Bruker spectrospin
spectrometer. CDCl3 (Sigma-Aldrich, Bangalore, India) was used as solvent and TMS as
an internal standard. FAB MS analyses were performed on a JEOL SX 102/Da-600
instrument equipped with direct inlet probe system. Column chromatography separations
were carried out on silica gel (Merck, 60–120 mesh, Mumbai, India). Precoated silica gel
plates (Merck, Silica gel 60 F254) were used for analytical thin layer chromatography
visualised by exposure to iodine and UV radiations.
3.2. Plant material
The stem bark of M. alba L. was collected from the campus of Jamia Hamdard,
New Delhi, and identified by Dr. H.B. Singh, Scientist F and Head, Raw Materials
Herbarium and Museum, National Institute of Science Communication and Information
Resources (NISCAIR), New Delhi. A voucher specimen of drug was deposited in the
herbarium of NISCAIR with a reference number NISCAIR/RHMD/Consult/-2008-09/
1059/90.
3.3. Extraction and isolation
The air-dried bark (2 kg) of M. alba were coarsely powdered and extracted exhaustively with
methanol using Soxhlet apparatus for 72 h. The extract was concentrated under reduced
pressure to get dark brown mass (260 g). Small portion of the extract was analysed chemi-
cally to determine the presence of different chemical constituents. The extract was dissolved
in minimum amount of methanol and adsorbed on silica gel (60–120 mesh) for preparation
of slurry. The slurry was dried and subjected to silica gel column chromatography.
3.3.1. �-Amyrin acetate (1)
Elution of the column with petroleum ether-chloroform (1:1) yielded colourless crystals of
1, recrystallised from acetone, (497 mg, 0.62% yield). Rf 0.69 (petroleum ether-chloroform

Natural Product Research
529
(1:1), melting point (m.p.) 225–226�C; þve ion FAB MS m/z (rel. int.): 468 [M]þ
(C32H52O2) (88.3).
3.3.2. Moruslanosteryl acetate (2)
Further elution of the column with petroleum ether-chloroform (1:1) furnished colourless
needles of 2, recrystallised from acetone, (122 mg, 0.15% yield). Rf 0.82 (petroleum ether-
chloroform, 1:1); m.p. 124–125�C; UV �max (MeOH): 257 nm (log " 4.2); IR �max (KBr):
1
2950, 2884, 1730, 1646, 1454, 1365, 1245, 1024 cm�1; H NMR (CDCl3): � 5.22 (1H, m,
H-6), 5.07 (1H, m, H-24), 4.51 (1H, dd, J¼5.5, 9.5 Hz, H-3a), 2.01 (3H, brs, OCH3), 1.65
(3H, brs, Me-26), 1.57 (3H, brs, Me-27), 1.10 (3H, brs, Me-19), 0.96 (3H, d, J¼6.3 Hz,
Me-21), 0.90 (3H, brs, Me-28), 0.82 (3H, brs, Me-29), 0.77 (3H, brs, Me-30), 0.73 (3H, brs,
Me-18); 13C NMR (CDCl3): � 37.79 (C-1), 29.66 (C-2), 81.08 (C-3), 43.48 (C-4), 145.94
(C-5), 125.09 (C-6), 33.73 (C-7), 33.90 (C-8), 50.73 (C-9), 36.79 (C-10), 22.01 (C-11), 35.73
(C-12), 48.77 (C-13), 51.24 (C-14), 24.16 (C-15), 27.53 (C-16), 53.20 (C-17), 13.09 (C-18),
18.54 (C-19), 35.12 (C-20), 18.11 (C-21), 24.77 (C-22), 27.28 (C-23), 117.55 (C-24), 130.86
(C-25), 25.68 (C-26), 25.31 (C-27), 25.82 (C-28), 23.72 (C-29), 19.51 (C-30), 170.81, 21.26
(OCOCH3); þve ion FAB MS m/z (rel. int.): 468 [M]þ (C32H52O2) (69.5), 453 (86.3), 410
(71.8), 408 (53.8), 393 (33.1), 378 (10.2), 357 (12.6), 342 (21.0), 315 (11.6), 297 (29.8), 282
(13.2), 267 (15.3), 208 (26.7), 202 (26.3), 194 (31.5), 188 (73.2), 174 (65.2), 148 (78.2), 144
(84.1), 142 (65.3), 134 (89.8), 119 (83.8), 111 (26.3), 95 (100).
3.3.3. �-Amyrin-�-D-glucopyranoside (3)
Elution of the column with chloroform-methanol (97:3) afforded colourless crystals of 3,
recrystallised from methanol, (705 mg, 0.88% yield). Rf 0.38 (chloroform-methanol, 24 : 1);
m.p. 210–215�C; þve ion FAB MS m/z (rel. int.): 588 [M]þ (C36H60O6) (2.6).
3.3.4. Moruslupenoic acid A (4)
Further elution of the column with chloroform-methanol (97:3) gave colourless
amorphous powder of 4, recrystallised from methanol, (171 mg, 0.21% yield). Rf 0.56
(chloroform-methanol, 24:1); m.p. 275–276�C; UV �max (MeOH): 253 nm (log " 4.1); IR
�
max (KBr): 3460, 2940, 2868, 1686, 1642, 1455, 1272, 1236, 1083 cm�1; 1H NMR (CDCl3):
� 4.68 (1H, brs, H2-29a), 4.56 (1H, brs, H2-29b), 3.50 (1H, dd, J¼5.5, 9.3 Hz, H-3a), 1.65
(3H, brs, Me-30), 1.24 (3H, brs, Me-23), 0.93 (3H, brs, Me-24), 0.88 (3H, brs, Me-25), 0.76
(3H, brs, Me-26), 0.65 (3H, brs, Me-28); 13C NMR (CDCl3): � 38.06 (C-1), 27.50 (C-2),
77.38 (C-3), 39.26 (C-4), 55.86 (C-5), 18.39 (C-6), 34.40 (C-7), 40.65 (C-8), 50.54 (C-9),
36.17 (C-10), 19.11 (C-11), 25.53 (C-12), 38.97 (C-13), 55.38 (C-14), 30.57 (C-15), 32.19 (C-
16), 42.41 (C-17), 47.02 (C-18), 49.05 (C-19), 150.67 (C-20), 29.63 (C-21), 36.84 (C-22),
28.47 (C-23), 14.79 (C-24), 16.14 (C-25), 16.31 (C-26), 177.65 (C-27), 16.89 (C-28), 109.93
(C-29), 19.36 (C-30); þve ion FAB MS m/z (rel. int.): 456 [M]þ (C30H48O3) (18.3), 441
(23.1), 438 (39.0), 426 (11.2), 411 (19.6), 397 (20.5), 393 (13.5), 378 (15.3), 306 (23.0), 248
(18.2), 234 (21.6), 220 (19.1), 208 (21.3), 168 (18.2), 154 (93.6), 150 (40.1), 140 (39.8), 136
(65.7), 122 (68.5).
3.3.5. Moruslupenoic acid B (5)
Further elution of the column with chloroform-methanol (97:3) gave colourless flakes of 5,
recrystallised from methanol, (445 mg, 0.55% yield). Rf 0.56 (chloroform-methanol, 24:1);
m.p. 275–276�C; UV �max (MeOH): 253, 280 nm (log " 5.3, 1.2); IR �max (KBr): 3436, 3390,
2927, 2855, 1686, 1638, 1457, 1375, 1237, 1106, 1032, 883 cm�1; 1H NMR (CDCl3): � 5.33
(1H, dd, J¼5.4, 6.0 Hz, H-12), 4.71 (1H, brs, H2-29a), 4.67 (3H, brs, H2-29b), 3.27 (1H, dd,

530
A. Ali and M. Ali
J¼5.4, 10.4 Hz, H-3a), 1.67 (3H, brs, Me-30), 1.20 (3H, brs, Me-23), 0.96 (3H, brs, Me-24),
0.91 (3H, brs, Me-25), 0.79 (3H, brs, Me-27), 0.74 (3H, brs, Me-28); 13C NMR (CDCl3):
� 38.37 (C-1), 27.62 (C-2), 77.98 (C-3), 38.89 (C-4), 55.61 (C-5), 20.42 (C-6), 33.90 (C-7),
54.92 (C-8), 50.07 (C-9), 37.74 (C-10), 26.88 (C-11), 132.51 (C-12), 146.76 (C-13), 40.27
(C-14), 30.12 (C-15), 31.85 (C-16), 41.95 (C-17), 48.74 (C-18), 46.43 (C-19), 150.32 (C-20),
29.14 (C-21), 36.69 (C-22), 28.62 (C-23), 15.62 (C-24), 17.85 (C-25), 177.97 (C-26), 15.08
(C-27), 14.21 (C-28), 108.89 (C-29), 19.23 (C-30); þve ion FAB MS m/z (rel. int.): 454 [M]þ
(C30H46O3) (15.3), 409 (38.7), 394 (31.5), 379 (18.6), 364 (18.3), 238 (25.0), 220 (21.6), 216
(31.9), 205 (26.3), 201 (29.8), 193 (16.1), 190 (18.3), 189 (39.0), 186 (30.5), 180 (21.7), 175
(36.2), 160 (45.8), 159 (37.2), 145 (63.8), 118 (81.2).
3.3.6. Betulinic acid (6)
Elution of the column with chloroform-methanol (89:1) produced colourless crystalline
mass of 6, recrystallised from chloroform-methanol (1:1), (346 mg, 0.43% yield). Rf 0.89
(chloroform-methanol, 7.3:1); m.p 113–115�C; þve ion FAB MS m/z (rel. int.): 457 [M]þ
(C30H49O3) (64.8).
4. Conclusions
This work characterised chemical compounds in the stem bark of M. alba that can be of
great help for its standardisation, as it is a drug of controversial identity in the traditional
system of medicine in India.
Acknowledgement
The authors are thankful to the Head, SAIF, Central Drug Research Institute, Lucknow for
recording spectral data.
References
Ali, M. (2001). Techniques in terpenoid identification. Delhi: Birla Publication.
Ali, M., Ravinder, E., & Ranachandram, R. (2000). New ursane-type triterpenic esters from the stem bark of
Thevetia peruviana. Pharmazie, 55, 385–389.
Anonymous (2001). The wealth of India: A dictionary of Indian raw materials and industrial products (Vol. 6,
pp. 429–437). New Delhi: CSIR.
Ansari, S.H., Ali, M., & Qadary, J.S. (1993). Three new tetracyclic triterpenoids from galls of Pistacia integrima
stem. Pharmazie, 48, 215–217.
Ayer, W.A., Flanagan, R.J., & Reffstrup, T. (1984). Metabolites of bird’s nest fungi-19: new triterpenoid
carboxylic acids from Cyathus striatus and Cyathus pygmaeus. Tetrahedron, 40, 2069–2082.
Boszormenyi, A., Szarka, Sz., Hethelyi, E., Gyurjan, I., Laszlo, M., Simandi, B., . . . Lemberkovics, E. (2009).
Triterpenes in traditional and supercritical-fluid extracts of Morus alba leaf and stem bark. Acta
Chromatographica, 21, 659–669.
Butt, M.S., Nazir, A., Sultan, M.T., & Schroen, K. (2008). Morus alba L. nature’s functional tonic. Trends in
Food Science and Technology, 19, 505–512.
Deshpande, V.H., Parthasarathy, P.C., & Venkataraman, K. (1968). Four analogues of artocarpin and
cycloartocarpin from Morus alba. Tetrahedron letters, 9, 1715–1719.
Ercisli, S., & Orhan, E. (2007). Chemical composition of white (Morus alba), red (Morus rubra) and black (Morus
nigra) mulberry fruits. Food Chemistry, 103, 1380–1384.
Ghosh, P., Mandal, A., Chakraborty, P., Rasul, M.G., Chakraborty, M., & Saha, A. (2010). Triterpenoids from
Psidium guajava with biocidal activity. Indian Journal of Pharmaceutical Sciences, 72, 504–507.
Li, S., Shi, Y., Shang, X.Y., Cui, B.S., Yuan, Y., Chen, X.G., . . . Shi, J.G. (2009). Triterpenoids from the roots of
Pterospermum heterophyllum Hance. Journal of Asian Natural Product Research, 11, 652–657.
Mahato, S.B., & Kundu, A.P. (1994). 13C NMR Spectra of pentacyclic triterpenoids – a compilation and some
salient features. Phytochemistry, 37, 1517–1575.
Malik, I., Ahmad, V.U., Anjum, S., & Basha, F.Z. (2010). A pentacyclic triterpene from Litchi chinensis. Natural
Product communication, 5, 529–530.

Natural Product Research
531
Mhaskar, K.S., Blatter, E., & Caius, J.F. (2000). Kiritikar and Basu illustrated: Indian Medicinal Plants, their
usage in Ayurveda and Unani medicines (Vol. 10, pp. 3185–3187). Delhi: Sri Satguru Publications.
Rama Rao, A.V., Deshpande, V.H., & Shastri, R.K. (1983). Structures of albanols A and B, two novel phenols
from Morus alba bark. Tetrahedron Letters, 24, 3013–3016.
Sharma, S.K., & Ali, M. (1996). New ��-lanostane-type triterpenic acid and 13, 14-seco-steroidal esters from the
roots of Artimisia scoparia. Journal of Natural Product, 59, 181–184.
Sirat, H.M., Susanti, D., Ahmad, F., Takayama, H., & Kitajima, M. (2010). Amides, triterpene and flavonoids
from the leaves of Melastoma malabathricum L. Journal of Natural Medicines, 64, 492–495.
Wang, X., Liu, K., & Xu, H. (2009). Studies on chemical constituents of aerial roots of Ficus microcarpa.
Zhongguo Zhong Yao Za Zhi, 34, 169–171.