Triterpenoids and triterpenoid saponins from the aerial parts of Fagonia indica Burm

Article abstract of DOI:10.1016/j.phytol.2015.07.001

Abstract As part of our search of new bioactive compounds from indigenous medicinal plants, phytochemical investigation of the aerial parts of Fagonia indica Burm led to the isolation of seven compounds including two new compounds, namely, indicacin (1) and fagonicin (2), and five known compounds (3-7) from the methanol extract. Compounds 6 and 7 are hitherto unreported from this plant. The structures of the new compounds were elucidated from their spectral data, mainly HREIMS, 1D NMR (¹H, ¹³C NMR, and DEPT) and 2D NMR (COSY, NOESY, HSQC and HMBC), and by comparison with the literature data. The new compounds 1 and 2 were assayed for their cytotoxicity against human colorectal cancer cell line H-29. Compound 1 exhibited 51.40% cytotoxicity at 6.25 μM/mL dose whereas compound 2 demonstrated 39.3% cytotoxicity at the same dose.

Full text of DOI:10.1016/j.phytol.2015.07.001

Phytochemistry Letters 13 (2015) 256–261
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Triterpenoids and triterpenoid saponins from the aerial parts of Fagonia
indica Burm
Rabia Farheen , Bina Shaheen Siddiqui *, Iffat Mahmood , Shabana Usman Simjeeb,c,
Saba Majeed
a
a
b,
a
b
Department of Chemistry, Federal Urdu University of Arts, Science and Technology, Gulshan-e-Iqbal, Karachi 75270, Pakistan
HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi
b
c
7
5270, Pakistan
A R T I C L E I N F O
A B S T R A C T
Article history:
As part of our search of new bioactive compounds from indigenous medicinal plants, phytochemical
investigation of the aerial parts of Fagonia indica Burm led to the isolation of seven compounds including
two new compounds, namely, indicacin (1) and fagonicin (2), and five known compounds (3–7) from the
methanol extract. Compounds 6 and 7 are hitherto unreported from this plant. The structures of the new
compounds were elucidated from their spectral data, mainly HREIMS, 1D NMR ( H, C NMR, and DEPT)
and 2D NMR (COSY, NOESY, HSQC and HMBC), and by comparison with the literature data. The new
compounds 1 and 2 were assayed for their cytotoxicity against human colorectal cancer cell line H-29.
Received 18 March 2015
Received in revised form 29 June 2015
Accepted 1 July 2015
Available online xxx
1
13
Keywords:
Fagonia indica
Leaves
Twigs and fruits
Triterpenes
Compound 1 exhibited 51.40% cytotoxicity at 6.25
9.3% cytotoxicity at the same dose.
mM/mL dose whereas compound 2 demonstrated
3
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Triterpenoid saponins
Cytotoxicity
1. Introduction
Several triterpenes and saponins have been reported from this
genus which include nahagenin, betulic acid (Rahman et al., 1982),
Fagonia indica Burm belongs to the family Zygophyllaceae
Pullaiah, 2006). It is commonly known as ‘sachchi booti’ in
Pakistan. It is a small genus of erect or prostrates, more or less
woody herbs and under shrubs (Manjunath, 1956). All species of
Fagonia are shrub, sherbets or herbs, rarely higher than (60–
hederagenin and ursolic acid (Rahman et al., 1984), 3
dihydroxy-23,28-di-O- -glucopyranosyl-taraxer-20-en-28-oic
acid, 23,28-di-O-3 -O- -glucopyranosyl-23-hydroxy taraxer-20-
en-28 oic acid (Ansari et al., 1987), 21, 22 -epoxy-23-O-
glucopyranosyl-nahagenin (Ansari et al., 1988), 3-sulfate esters of
,27-dihydroxyolean-12-en-28-oic acid and 3 -27-dihydroxy-
28-carboxy-O- -glucopyranosylolean-12-en (Perrone et al.,
2007). In the present article, we report on the isolation and
structure elucidation of two new compounds, a triterpenoid
glycoside (1), a triterpene (2) and five known compounds (3–7)
from the methanol extract of the aerial parts of Fagonia indica
Burm. Compounds 5 and 6 are reported for the first time from this
plant.
b, 23-
(
b-D
D
b
a
b-D-
1
00) cm, and up to about 100 cm wide. The genus Fagonia is
confined to warm and arid areas of all continents except Australia
Beier, 2005). It is found mostly in Indo-Pakistan subcontinent
3
b
b
b-D
(
westwards to North and East tropical Africa in arid and semi arid
region (Shinwari and Shah, 2003). In Pakistan it is represented by 8
genera and 22 species (Ghafoor, 1974). The plant has been used by
local people in the treatment of fever, asthma, vomiting, dysentery,
urinary discharge, leucoderma, biliousness and typhoid and as
blood purifier and plant ash is given to children suffering from
anemia. The fruit of this plant is rich in ascorbic acid (Khare 2007).
The twigs are commonly applied as tooth brushes and bark in the
scabies (Shinwari and Shah, 2003). Aerial parts of Fagonia indica
Burm are used as a remedy for tumors, and leaf and twigs are used
for cancer (Graham et al., 2000).
2. Results and discussion
Aerials parts of Fagonia indica were repeatedly extracted with
MeOH by soaking the plant material in methanol at room
temperature. The methanol extract was shaken out with ethyl
acetate and water. After usual workup the residue obtained from
ethyl acetate fraction was subjected to various chromatographic
techniques to afford compounds 5–7. The aqueous phase was
treated with n-butanol and the butanol fraction afforded
*
Corresponding author. Fax: +92 21 99261713.
E-mail address: siddiqui_bina@yahoo.com (B.S. Siddiqui).
http://dx.doi.org/10.1016/j.phytol.2015.07.001
874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1

R. Farheen et al. / Phytochemistry Letters 13 (2015) 256–261
257
compounds 1–4 after repeated reverse phase column and thick
layer chromatography (Fig. 1).
Compound 1 was isolated as white amorphous powder. Its
molecular formula was determined as C36 10 on the basis of
HRFABMS (negative mode) peak of [M ꢀ H] at m/z 647.3802
56
H O
+
Known compounds were identified as 3
syl-20-en-23-hydroxytaraxer-28-oic acid (3) and 3
O- -glucopyranosyl-28-carboxy-O- -glucopyranosyl-tar-
axer-20-en (4) (Akbar et al., 1987), -amyrin (5) (Mahato and
Kundu, 1994), lupeol (6) (Mahato and Kundu, 1994; Burns et al.,
000), and -sitosterol (7) (Zhang et al., 2006) respectively, by
b-O-b
-D-glucopyrano-
+
b
-hydroxy-23-
[M ꢀ H] (calc. 647.3780 for C36
HREIMS at m/z 468.6736 (C30
spectral data. Its IR spectrum showed absorptions for hydroxyl
H
55
O
10) from the HRFABMS and
+
13
b-
D
b-
D
H
44
O
4
; M -glucose) and C NMR
b
ꢀ
1
ꢀ1
(3425 cm ), carbonyl (1730 cm ), carboxyl (1710) and olefin
ꢀ
1
1
2
b
(1658 cm ). The H NMR spectrum showed five methyl singlets at
0.74, 0.81, 0.85, 1.15 and 1.26 connected to carbons at 16.4, 18.1,
16.2, 26.7 and 28.3 respectively in the HSQC spectrum, a triplet for
H-12 of pentacyclic triterpenes at 5.20 (1H, t, J = 3.6 Hz)
connected to C-12 at 122.8 and a double doublet at 2.81
(dd, J = 13.0, 4.5) for H-18 connected to C-18 at 42.7. These data
1
13
comparison of their mass, H and C NMR spectral data with the
reported data.
d
d
The new compounds (1) and (2) were assayed for their
cytotoxicity against human colorectal cancer cell line H-29 and
d
H
d
C
d
H
showed 51.40% and 48.3% inhibition at 6.25 and 12.5
concentration respectively.
m
M/mL
d
C
were suggestive of an oleanane type skeleton of 1. Further, two
2
9
30
OH
1
9
20 21
H
1
2
1
3
17
22
1
1
2
5
26
14
18
COOH
28
CHO
H
1
9
16
1
5
2
1
0
8
CH OH
2
3
7
4
27
6
O
5
HO
CH O-β-D-Glcp
2
2
4
23
1
2
COOH
C
O
O-D-Glcp
β-D-GlcpO
HO
CH OH
CH O-β-D-Glcp
2
2
3
4
HO
HO
5
6
HO
7
Fig. 1. Structures of 7 compounds isolated from Fagonia indica.

2
58
R. Farheen et al. / Phytochemistry Letters 13 (2015) 256–261
pairs of downfield methylene protons at
H-23a/b) and 3.24, 3.55 (d, J = 11.0 Hz, H-27a/b) showing HSQC
correlation with C-23 ( 66.4) and C-27 ( 76.4) revealed that two
methyls of the primary skeleton have been functionalized to a
hydroxymethylene group (C-27) and a oxymethylene (C-23). In
addition, the hydroxyl group at C-3 was not present and instead a
d
H
3.40, 3.62 (d, J = 10.8 Hz,
(J = 6.0 Hz). Accordingly, it was indicated that 2 is a taraxarane or
d
H
ursane triterpenoid. The presence of only one methyl doublet
further indicated a substituent at either C-19 or C-20. The
molecular formula showed index of hydrogen deficiency as six,
justifying a pentacyclic triterpenoid skeleton and one aldehyde
d
d
ꢀ
1
1
group as indicated by the IR (
n
max 2730, 1715 cm ) as well as H
9.51, s, 206.7). Two downfield carbons
d 73.2 (C) (Table 2) showed the presence of two
13
carbonyl group was indicated at
group at C-23 was deduced by the chemical shifts of C-23 and C-24
Table 1) (Mahato and Kundu, 1994) and the location of hydroxyl
d
C
211.7. The location of a hydroxyl
and C NMR spectra (
at 78.9 (CH) and
hydroxyl groups indicated by the IR spectrum (
The secondary hydroxyl group was placed at C-3 on biogenetic
consideration and its -disposition was decided from the double
doublet of H-3 ( 3.14, J = 11.2, 4.8 Hz). The aldehyde proton ( 9.51)
showed HMBC interaction with C-17 ( 61.5) and C-18 ( 49.3), thus
d
H
d
C
d
ꢀ
1
(
nmax 3384 cm ).
group at C-27 and of the carbonyl group at C-3 was assigned by
HMBC correlations of H-27 with C-13 and C-14 and of both H-23
and H-24 with C-3. The 1H and C NMR data (Table 1) further
b
13
d
d
revealed a glucose moiety by an anomeric proton at
J = 7.5 Hz, H-1 ) connected to the anomeric carbon at
d
d
H
4.45 (1H, d,
102.8 (C-1 )
d
d
0
0
C
indicating the aldehyde group to be located at C-28. In order to
decide between the ursane or taraxarane skeleton and location of
second OH group at C-19 or C-20, the carbon-13 NMR data of C-13,
C-18, C-29 and C-30 of 2 were compared with several examples of
similar structures in the literature and were found closer to those
of taraxarane skeleton. In both 19- and 20-hydroxy ursane, C-13
in the HSQC spectrum, oxygen bearing methylene protons at
d
3.79
0
(
1H, dd, J = 11.7, 4.2 Hz, H-6 a) and
d
3.92 (1H, dd J = 11.7, 2.7 Hz,
0
0
H-6 b) connected to a CH
2
carbon at
d
62.4 (C-6 ) and four methine
protons at
carbons at
d
d
4.14, 3.85, 4.22 and 3.20 (each 1H, m) connected to CH
81.1, 75.4, 77.4, 76.1 in the HSQC spectrum. Both H-23a
3
and H-23b showed J connectivity in the HMBC spectrum with the
anomeric carbon at
carbon at
appears downfield at
(Kuo et al., 2014) respectively while in taraxarane having a
hydroxyl or oxygen substituent at C-20, C-13 shift appears at 38.9
(Susunaga et al., 2001) and 39.3 (Zhao et al., 2006) which were
closer to that of 2 ( 38.1) thus suggesting it to be a 20-hydroxy
d 50.6 (Misra and Laatsch, 2000) and d 49.4
0
d
C
102.8 (C-1 ) and an up field quaternary
d
48.0 (C-4). Hence glucose was placed at C-23. The
d
0
0
0
0
0
0
connectivity of H-1 with H-2 ; of H-2 with H-3 ; of H-3 with H-4 ;
of H-4 with H-5 ; and of H-5 with H-6 in the 1H– H COSY
0
0
0
0
1
d
spectrum led to assign each proton of the sugar moiety. The NMR
profile of sugar protons and carbons showed that glucose was b-D-
pyranose. The NOESY correlations between H-5, H-9 and H-23a/b
and between H-9 and H-27a/b supported the assignment of H-23
and H-27, and their respective carbons through HSQC interactions.
Thus the structure of indicacin was determined as 3-oxo-12-en-23-
taraxarane triterpene. The NOESY interactions between H-18 and
H-27, H-29 as well as between H-13 and H-19, H-26 further
supported that ring-D and -E are trans-fused. Chemical shifts for
C-20 (
C-20:73.6;
C-20:27.4) (Susunaga et al., 2001) geometry suggesting that the
hydroxyl group at C-20 is and hence 2 has S configuration at C-20.
d
73.2) and C-30 (
d
31.2) of 2 compare well with 20S
(d
d
C-30: 30.3) geometry as against 20R (
d
C-20: 75.3;
d
O-b
-D-glucopyranosyl-27-hydroxyolean-28-oic acid. The sugar
b
moiety was further confirmed as
of 1 followed by GC analysis of the ester derivative and comparison
with authentic sample.
D
-glucose through acid hydrolysis
Similar analogies were noted for C-18 shifts which are in
agreement with a 20S-hydroxytaraxastane. A relatively downfield
shift of C-17 may be attributed to hydrogen bonding between OH at
Compound 2 was obtained as amorphous powder. Its molecular
mass was determined on the basis of HREIMS at m/z 458.7222
C-20 and CHO at C-17 and a d effect of OH on C-17 (Eggert et al.,
1976). The configuration of various centers and assignment of
methyl groups was determined through NOESY interactions
between H-24, H-25 and H-26; of H-23 with H-3 and H-27 as
well as of CHO with C-26. Hence the structure of (2) was defined as
which was in accordance with its molecular formula C30
50 3
H O .
Analysis of its NMR data indicated that it is a pentacyclic triterpene
13
of ursane skeleton (Seebacher et al., 2003). Thus C NMR (broad
ꢁ
ꢁ
band, DEPT 90 and 135 ) showed 30 carbons including 7 methyls,
1
3
b
, 20S-dihydroxytaraxastane-28-al.
1
0 methylenes, 7 methines and 6 quarternary carbons. The H NMR
The NMR data of compounds 3 and 4 (Table 2) matched well
spectrum revealed the presence of six methyl singlets at
.80, 0.87, 0.94, 0.96 and 1.12 and one methyl doublet at
d
d
0.73,
1.22
with those reported in literature (Ansari et al., 1987). However this
report presents a complete assignment of all the protons and
0
Table 1
1
H (600 MHz) and 13C (150 MHz) NMR data of compound 1 in MeOH-d4 (
d
, ppm; J, Hz).
Position
Position
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
/CH
/CH
/C
2
39.2
33.7
1.26 (m), 1.47 (m)
2.13 (m), 2.61 (m)
19/CH
20/C
21/CH
22/CH
23/CH
24/CH
25/CH
26/CH
27/CH
28/C
2
43.2
30.7
30.6
30.2
66.4
16.4
16.2
18.1
76.4
1.60 (m), 0.85 (m)
–
2
211.7
48.0
49.0
19.2
33.5
39.0
46.0
37.9
24.4
122.8
146.0
40.4
27.0
24.4
47.0
–
2
1.46 (m), 1.15 (m)
2.01 (m), 1.50 (m)
3.40 (d, 10.8), 3.62 (d, 10.8)
0.74 (s)
0.85 (s)
0.81 (s)
3.24 (d, 11.0), 3.55 (d, 11.0)
–
/C
–
2
2
3
3
3
2
/CH
/CH
/CH
/C
1.66 (m)
1.43 (m), 1.61 (m)
1.25 (m), 1.27 (m)
–
2
2
/CH
1.47 (m)
–
1
0/C
1/CH
2/CH
3/C
4/C
5/CH
6/CH
7/C
8/CH
177.1
28.3
26.7
1
1
2
1.88 (m), 1.73 (m)
5.20 (t, 3.6)
29/CH
30/CH
3
1.26 (s)
1.15 (s)
3
1
1
–
Glucose:
0
–
1 /CH
102.8
81.1
75.4
77.4
76.1
62.4
4.45 (d, 7.5)
4.14 (m)
3.85 (m)
4.22 (m)
0
1
1
2
2
1.15 (m), 1.54 (m)
1.88 (m), 1.01 (m)
–
2 /CH
0
3 /CH
0
1
1
4 /CH
0
42.7
2.81 (dd, 13.0, 4.5)
5 /CH
3.20 (m)
0
6
/CH
2
3.79 (dd, 11.7, 4.2), 3.92 (dd, 11.7, 2.7)
Assignments based on HSQC, HMBC and DEPT experiments.

R. Farheen et al. / Phytochemistry Letters 13 (2015) 256–261
259
Table 2
with a Jeol, JMS-600H mass Spectrometer operating in EI mode
with ion source at 250 C and electron energy at 70 eV. Carrier gas
1
H (500 MHz) and 13C (125 MHz) NMR data of compound 2 in CHCl
3
(d
, ppm: J, Hz).
ꢁ
Position
d
C
d
H
Position
d
C
d
H
was helium at a pressure of 17.2 psi. All fractions were dissolved in
chloroform and injection volume was adjusted between 1.0 and
5.0 mL depending upon the detector response.
1
2
3
4
5
6
7
8
9
/CH
/CH
/CH
/C
/CH
/CH
/CH
/C
2
2
38.6 0.87 (m), 1.68 (d, 3.2) 16/CH
28.1 0.94 (m), 0.96 (m)
78.9 3.14 (dd, 11.2, 4.8)
38.8
55.1 0.67 (m)
18.2 1.34 (m), 1.48 (m)
33.0 1.24 (m), 1.58 (m)
41.2
50.3 2.19 (m)
37.0
21.2 1.21 (m), 1.47 (m)
29.2 0.94 (m), 1.23 (m)
38.1 2.02 (m)
2
27.3 1.61 (m), 1.53 (m)
61.5
17/C
18/CH
19/CH
–
49.3 2.18 (dd, 10.8)
46.8 1.68 (m)
73.2
29.5 1.22 (m) 1.40 (m)
34.5 2.01 (m), 1.50 (m)
27.9 0.94
15.3 0.73 (s)
16.1 0.80 (s)
16.2 0.87 (s)
14.5 0.96
–
20/CH
3.3. Plant material
2
2
21/CH
22/CH
23/CH
24/CH
25/CH
26/CH
27/CH
28/CH
29/CH
30/CH
2
2
3
3
3
3
3
The aerial parts of Fagonia indica Burm were collected from
Hyderabad, Sindh region during the month of August to Septem-
ber. The plant was authenticated by Afshen Ather, Curator of the
Centre for Plant Conservation, Department of Botany, University of
Karachi and a voucher specimen (no. 86583) was deposited in the
Herbarium of the same department.
–
/CH
1
1
1
1
1
1
0/C
1/CH
2/CH
3/C
4/C
5/CH
–
2
206.7 9.51
43.4
–
3
3
25.0 1.22 (d, 6.0)
31.2 1.12 (s)
2
29.0 0.95 (m), 1.22 (m)
Assignments based on HSQC, HMBC and DEPT experiments.
3.4. Extraction and isolation
The aerial parts (5 kg) of Fagonia indica were repeatedly (ꢂ3)
extracted with methanol (12 L each time) at room temperature.
The extract obtained on evaporation of solvent under reduced
pressure was partitioned between EtOAc and water. The EtOAc
layer was treated with anhyd. Na SO , charcoaled and concentrat-
ed. A part (2 g) of this residue (60 g) on silica gel CC and elution
with petrol-EtOAc (9:1) afforded a mixture of compounds which
were purified on repeated silica gel CC using mixture of petrol-
EtOAc to afford 5 (9.5:0.5; 32.3 mg), 6 (9.25: 0.75; 25.6 mg) and 7
(9:1; 15 mg).
carbons on the basis of 2D NMR analysis. Compounds 5, 6 and 7
were identified by matching their NMR data with the reported
values as
b
-amyrin (Mahato and Kundu, 1994), lupeol (Burns et al.,
-sitosterol (Zhang et al., 2006).
2
4
2
3
3
000) and
b
. Experimental
.1. General experiment
The main aqueous phase was concentrated under reduced
pressure and treated with n-BuOH. The n-butanolic phase was
washed, dried over anhydrous Na SO and concentrated under
Infrared spectra were measured on VECTOR 22 spectropho-
ꢀ
1
tometer;
n in cm . Specific rotations were determined using
2
4
JASCO P-200 polarimeter. NMR spectra were measured on Avance
vacuum to give a gummy residue (125 g). This was separated by
reverse phase column chromatography (LH-20; H O, H O-MeOH
gradient system, 1:1 to 2:8) to afford 3 major fractions (LH-1, LH-2
AV-300, and AV-500 and AV-600 spectrometers operating at 300,
2
2
1
5
00 and 600 MHz for H NMR and 75, 125 and 150 MHz
13
respectively for C NMR spectra. EI and HREIMS were recorded
on MSRoute mass spectrometer and Jeol-JMS-HX-110 instruments
respectively. Column chromatography (CC) was performed with
silica gel 60 PF254 (E. Merck, 70–230 mesh size). Aluminum cards
precoated (0.5 mm thickness) with silica gel SiF254 (E. Merck) and
RP-18 F254 S (E. Merck) were used for analytical chromatography.
Sephadex LH-20, (Sigma–Aldrich) and silica gel RP-18 (Merck)
were used for column chromatography of more polar fractions. For
TLC of polar compounds RP-18 F254 (E. Merck, 0.25 mm) precoatd
TLC glass plates were used. HPLC was carried out using preparative
recycling HPLC (JAIGEL-ODS-M80).
and LH-3). LH-1 (0.50 g) was further purified by reverse phase
column chromatography (RP-18 silica gel; H O-MeOH; 1:1) and
2
yielded 10 fractions. Fraction 1 (0.37 g) was reloaded on RP-18
column to give 15 fractions. Fraction no. 12 afforded an amorphous
solid pure on TLC. This was identified as 3 (28.0 mg). Fraction LH-2
(0.58 g) afforded 36 fractions. These were combined on the basis of
TLC to give 10 fractions. Fraction 10 was subjected to preparative
recycling HPLC (JAIGEL-ODS-M80; Serial 208,550, JAL LC-908W)
using MeOH-H O (60:40) as mobile phase; flow rate (3 mL/min) to
2
afford one pure compound characterized as a new compound 2
(18.0 mg). LH-3 (0.24 g) gave 10 fractions. These fractions were
combined on the basis of TLC to give 3 fractions. Fraction 2 was
3.2. Cancer cell line, chemicals and spectral measurements
further purified on RP-18 column (H O: MeOH; 1:1). It afforded a
2
pure compound characterized as a new constituent 1 (12.5 mg).
For anti-cancer activity, human colorectal cancer cell line HT 29
Fraction 3 was purified on RP-18 TLC cards using MeOH-H O to
2
was purchased from American Type Tissue Culture Collection
ATCC, USA). The cells were cultured in DMEM (Sigma Chemical,
MO, USA) supplemented with 1% penicillin and streptomycin, 1%,
amphoterecin B, 1% sodium pyruvate, 1% -glutamine and 10% fetal
bovine serum [FBS (Sigma Chemical, MO, USA)] in a humidified
afford 4 (8:2; 5.8 mg).
(
3.5. 3-Oxo-12-en-23-O-
acid (1) (=indicacin)
b-D-glucopyranosyl-27-hydroxyolean-28-oic
L
ꢁ
]D 24 + 11.5 (c, 0.3, MeOH);
HRFABMS m/z: 647.3802 [M ꢀ H] (calc. 647.3780 for C36H55O10).
ꢁ
2
atmosphere at 37 C containing 5% CO . Gas chromatography was
Colorless amorphous powder; [a
+
carried out using flame ionization detector (FID) on the less polar
1
+
capillary column OPTIMA -5-Accent (60m ꢂ 0.32 mm ID with
30 44 4 6 12 6 4
HREIMS m/z: 468.6736 (C H O ; M -C H O ; calcd for C30H44O ,
ꢀ1
0
.25
m
m film thickness of 5% phenyl/95% methyl silicone) installed
468.6752); IR (KBr) nmax cm : 3425, 3093, 2984, 1730, 1710, 1658.
1
13
on a Shimadzu GC-10 for GC-FID analysis. The analysis was
H and C NMR data are given in Table 1.
ꢁ
performed with an initial temperature 50 C for 2 min then ramped
ꢁ
ꢁ
at a rate of 7 C/min. to a final temperature of 260 C with holding
3.6. 3 , 20S-Dihydroxytaraxastane28-al (2) (=fagonicin)
b
time of 30 min. Injector with a splitting ratio of 1:8 was set at
ꢁ
ꢁ
24
ꢁ
235 C and FID at 400 C. Carrier and make up gas was nitrogen
Colorless amorphous powder; [
a
]D + 34.5 (c = 0.3, MeOH);
H50O3
with a flow of 12.334 mL/min. at a pressure of 90 kPa. For GC-MS
experiments Agilent 6890 gas chromatograph, equipped with ZB-
HREIMS m/z: 458.7222 (C30 C
H
50
O ; M+; calcd. for
3
30
1
ꢀ
1
3
458.7232); IR nmax (CHCl ) cm : 3384, 2933, 2730, 1715. H and
13
5MS (30m ꢂ 0.32 ID and 0.25
m
m film thickness) was combined
C NMR data are given in Table 2.

2
60
R. Farheen et al. / Phytochemistry Letters 13 (2015) 256–261
4 7
(C15H23) (57.2), 133.1027 (C10H13) (45.3), 55.0774 (C H ) (10.0); H
1
3
.7. 3
b-O-b
-
D
-Glucopyranosyl-20-en-23-hydroxytaraxer-28-oic acid
13
(
3)
and C NMR data are given in Table 3.
2
4
+200 (c, 0.3, MeOH);
Colorless amorphous powder; [
a
]
D
3.9. Acid hydrolysis of compound 1
+
HREIMS m/z (rel.int.): 634.8428 (C36
H
58
O
9
;
M ; calcd for
+
+
C
C
36
H
H
58
O
O
9
, 634.8488), 456.7060 (C30
H
48
O
3
; M -C
H
H
6 12
O
6
calcd for
Compound 1 (2 mg) was refluxed with HCl (0.5 N, 2 mL) for 2 h.
The hydrolyzed product was extracted with CH Cl . The residue
obtained on usual work up of the aqueous layer was dissolved in
pyridine (1 mL) and a solution of -cysteine ethyl ester hydrochlo-
+
30
48
3
, 456.7074) (5.0), 248.1766 (C16
13) (62.3), 55.0774 (C
and C NMR data are given in Table 3.
24
O
2
) (30.3), 203.1811
2
2
1
(
C
15
H
23) (65.2), 133.1027 (C10
H
4 7
H ) (33.2). H
1
3
L
ride (0.1 M) in pyridine (2 mL) was added. The reaction mixture was
ꢁ
3.8. 3
b-Hydroxy-23-O-
b
-D-glucopyranosyl-28-carboxy-O-
b-
D-
heated at 60 C for 1 h. Acetic anhydride (1 mL) was then added and
glucopyranosyl-taraxer-20-en (4)
the mixture was heated for another 1 h. Acetylated thiazolidine
derivative, obtained after drying the mixture in vacuum was
subjected to GC under following conditions: capillary column SPB-
5 (60 m ꢂ 0.32 mm, 0.25 lm); carrier gas N2; injection temperature
2
4
ꢁ
Colorless amorphous powder; [
a
]
578
+ 1 (c, 0.4; MeOH);
O
+
HREIMS m/z (rel.int.): 796.9970 (C42
H
68
14
;
M ; calcd for
; M -2(C ; calcd
) (23.3), 203.1811
+
+
ꢁ
ꢁ
ꢁ
C
42
H
68
O
14 , 796.9908), 436.6764 (C30
H
44
O
2
H
6 12
O
6
250 C; detection temperature 260 C; column temperature 150 C
+
ꢁ
ꢁ
for C30
H
44
O
2
, 436.6764) (28), 248.1766 (C16
H
24
O
2
(1 min), 10 C/min to 260 C (60 min). The configuration was
determined by comparing the retention time with acetylated
thiazolidine derivatives prepared in a similar manner from
Table 3
NMR data of compounds 3 ( H 500 and C 125 MHz) and 4 ( H 300 and 13C 75 MHz)
1
13
1
R
standard sugar (t D-glucose 24.04 min).
in MeOH-d4 (
d
, ppm; J, Hz).
Position
3
4
3.10. Determination of growth inhibition activity
d
C
d
H
d
C
d
H
3
.10.1. Compounds preparation
The compounds (each 1 mg/mL) were taken in sterile 0.01 N
1
2
3
4
5
6
7
8
9
/CH
/CH
/CH
/C
2
39.5
27.4
73.1
42.0
48.9
19.1
35.0
43.1
51.8
38.0
22.9
28.9
40.3
43.4
30.5
35.1
50.9
50.2
38.6
1.00 (m), 1.59 (m)
1.59 (m), 1.59 (m)
3.82 (dd,11.2, 4.5)
–
39.4
27.5
78.6
42.1
48.5
19.0
34.8
43.1
51.5
38.0
22.8
28.7
40.4
43.5
30.2
33.7
50.4
50.2
38.5
144.0
118.1
38.3
1.06 (m), 1.06 (m)
1.60 (m), 1.60 (m)
3.49 (dd, 11.2, 4.5)
–
2
NaOH to obtain the stock solutions. The working solutions were
prepared from the stock solution by diluting it in the Dulbecco
modified Eagle medium (DMEM). Five different working concen-
trations (Table 4) were used to treat the HT 29 cells for MTT assay.
The cells treated with only NaOH were used as control. Final
concentration of NaOH used in the assay was 0.01 N.
/CH
/CH
1.28 (d,11.0)
1.48 (m), 1.34 (m)
1.48 (m), 1.30 (m)
–
1.31 (d, 11.0)
1.47 (m), 1.31 (m)
1.47 (m), 1.31 (m)
–
2
/ CH
/C
/CH
2
1.45 (dd, 9.5,4.1)
–
1.47 (dd, 9.5, 4.1)
–
1
1
1
1
1
1
1
1
1
1
2
2
0/C
1/CH
2/CH
3/CH
4/C
5/CH
6/CH
7/C
8/CH
9/CH
0/C
1/CH
2/CH
2
1.30 (m), 1.21 (m)
1.20 (m), 1.59 (m)
2.30 (ddd)
1.60 (m), 1.21 (m)
1.20 (m), 1.68 (m)
2.30 (ddd)
–
3
.10.2. Microscopy
2
Cells were grown on 25 cm3 flasks and treated with test
fractions mentioned above or 0.01N of NaOH for 24 h. The Nikon
inverted microscope was used at 10ꢂ and 20ꢂ magnifications to
examine the effect of fractions of F. indica on growth inhibition of
HT 29 cells. MTTassay was used further to validate our observation.
–
2
2
1.12 (m), 1.48 (m)
1.48 (m), 1.88 (m)
–
1.12(m), 1.55(m)
1.43(m), 2.03(m)
–
1.21 (d, 10.8)
2.07 (m)
1.21 (d, 10.8)
2.07 (m)
143.6
118.8
39.8
–
–
3.10.3. MTT assay to evaluate growth inhibition
5.22 (br.d, 6.6)
1.59 (br. d,15.9),
5.22 (br.d, 6.6)
1.78 (br. d, 15.9)
2.27 (dd,15.9,7.2)
3.66 (d, 11.5),
3.44 (d,11.5)
0.69 (s)
0.87 (s)
0.94 (s)
0.99 (s)
–
The effect of compounds 1 and 2 on growth inhibition of
colorectal cancer cells was evaluated by the MTT assay which
calorimetrically quantifies insoluble purple formazan compound
produce by viable cells. Briefly, monolayer of the cells was
trypsinized, re-suspended in medium containing 10% FBS and
2
2
2
2
2
.27 (dd,15.9,7.2)
3.70 (d,10.0),
.42 (d,10.0)
3/CH
2
75.9
75.6
3
2
2
2
2
2
2
3
4/CH
5/CH
6/CH
7/CH
8/C
3
3
3
3
12.7
17.3
0.70 (s)
0.88 (s)
0.97 (s)
0.98 (s)
–
12.6
17.3
3
ꢂ10 cells/100
m
L were plated in 96-wells plate and kept in a
16.9
15.5
170.3
24.1
22.2
16.6
15.5
175.8
23.8
22.0
ꢁ
humidified atmosphere at 37 C containing 5% CO
when monolayer was established media was aspirated and 100 mL
2
. After 24 h
9/CH
0/CH
3
1.00 (d, 6.6)
1.59 (s)
1.00 (d, 6.6)
1.60 (s)
Table 4
3
Growth inhibitory effect indicacin (1) and fagonicin (2) isolated from F. indica on HT
Glu-1
29 cell line at different concentrations.
0
1
2
3
4
5
6
/CH
/CH
/CH
/CH
/CH
/CH
102.9
81.2
75.6
76.2
77.2
62.5
4.44 (d, 8.0)
4.15 (dd, 8.5,7.5)
3.83 (m)
3.44 (m)
4.22 (m)
102.9
81.1
75.7
76.1
77.1
62.4
4.44 (d, 7.5)
4.15 (m)
3.85 (m)
3.44 (m)
4.23 (m)
0
0
0
0
0
Compounds
Indicacin
Concentration (
m
M/mL)
% Growth inhibition
6.25
12.50
25.0
50.0
51.40404
48.31951
47.09734
29.75411
2
3.89 (dd,11.7,4.2)
3.78 (dd,11.7,4.2)
3.88 (dd,11.7,2.7)
3
.79 (dd,11.7,2.7)
Fagonicin
6.25
12.50
25.0
50.0
39.3569
48.37771
37.55274
34.35181
Glu-2
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
/CH
/CH
/CH
/CH
/CH
/CH
–
–
–
–
–
–
–
–
–
–
–
–
95.7
71.1
74.2
78.3
72.7
62.4
5.39 (d, 8.1)
3.33 (m)
3.25 (m)
3.38 (m)
3.83 (m)
Control (NaOH)
6.25
12.50
25.0
50.0
0.758
0.741
0.816
0.827
2
3.78 (dd,12.0,5.4)
3
.69 (dd,12.0,3.0)
Assignments based on HSQC, HMBC and DEPT experiments.
Average of three independent determinations values are mean ꢃ SD.

R. Farheen et al. / Phytochemistry Letters 13 (2015) 256–261
261
of various concentrations of fractions and test compounds of F.
indica in media containing 1% FBS were added. Maximum NaOH
concentration used was 0.01 N. Cells treated with 0.01% NaOH in
media (containing 1% FBS) were used as untreated controls. After
Eggert, H., VanAntwerp, C.L., Bhacca, N.S., Djerassi, C., 1976. Carbon-13 nuclear
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No. 76. National Herbarium, Agricultural Research Council, Rawalpindi,
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Graham, J.G., Quinn, M.L., Fabricant, D.S., Farnsworth, N.R., 2000. Plants used against
24 h, sample solution in the wells was flicked off and 100 mL of
cancer- an extension of the work of Jonathan Hartwell. J. Ethnopharmacol. 73,
0
.5 mg/mL MTT dye (Promega, USA) was added to each well and
3
47–377.
ꢁ
incubated in 37 C, 5% CO
2
for 3 h. After incubation, supernatant
Khare, C.P., 2007. Indian Medicinal Plants, an Illustrated Dictionary. Springer Verlag,
Berlin/Heidelberg, Germany p. 259.
was removed and 100
mL of DMSO was added to each well to
Kuo, P.-C., Chen, G.-F., Yang, M.-L., Lin, Y.-H., Peng, C.-C., 2014. Chemical constituents
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solubilize formazan. The absorbance was recorded on 96-wells
plate ELISA reader at 490 nm. The percentage of viable cells
following treatment was normalized to untreated controls. Assays
were performed at least three times in triplicates. The growth
inhibition (%) was calculated using the following formula:
13
Mahato, S.B., Kundu, A.P., 1994. C NMR spectra of pentacyclic triterpenoids—a
compilation and some salient features. Phytochemistry 37, 1517–1575.
Manjunath, B.L.,1956. The Wealth of India, vol. 4. Council of Scientific and Industrial
Research, New Dehli p. 01.
ꢀ
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Misra, L., Laatsch, H., 2000. Triterpenoids, essential oil and photo-oxidative 28-13-
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test reading
control reading
Growth inhibitionð%Þ ¼ 100 ꢀ
ꢂ 100
Rahman, A., Ansari, A.A., Steven, A., Clardy, J., 1982. The isolation and structure of
Appendix A. Supplementary data
nahagenin. Heterocycles 19, 217–220.
Rahman, A., Ansari, A.A., Lennart, K., 1984. Hederagenine, ursolic acid and pinatol
from Fagonia indica. J. Nat. Prod. 47, 186–187.
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