Supplement-1
Isolation, cytotoxic evaluation and simultaneous quantification of eight bioactive
secondary metabolites from Cicer microphyllum by high performance thin layer
chromatography†
Alamgir A. Dara, Santosh K. Ratha, Afnan Qaudrib, Sheikh A. Tasduqb, Anil Kumarc,
Payare L. Sangwana,*
a
Bioorganic Chemistry Division, bPK-PD Toxicology Division,
CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu Tawi- 180001, India
c
College of Sciences, Sri Mata Vaishno Devi University, Katra Jammu-182320, India
Running Title: Quantification and validation of cytotoxic markers in Cicer
microphyllum
† IIIM Publication no. IIIM/1542/2015
*Correspondence: Dr. PL Sangwan Scientist, E-mail: plsangwan@iiim.ac.in (PL Sangwan)
Tel.: +91-191-2569024; fax: +91-191-2569333
Contents
Page No.
2.3. Preparation of extracts and isolation of the chemical constituents 2-3
1H
NMR, 13C NMR, MS SPECTRA
4-11
Figure S1
12
Tables S1-S2
13-14
References
15
1
2.3. Preparation of extracts and isolation of the chemical constituents
The DCM: MeOH extract of C. microphyllum was prepared by exhaustive maceration
of the dry and pulverised plant material (2.0 kg) with mixture of DCM: MeOH (1:1) three
times. The extract was then evaporated to dryness in a rotary evaporator under reduced
pressure at 35oC to afford a dark brown viscous liquid 180.0 g with extractive value (EV)
9.0%. The crude DCM: MeOH extract (150 g) was suspended in water and partitioned with
petroleum ether, chloroform, and methanol to yield three fractions. All three fractions were
screened by TLC and maximum numbers of secondary metabolites were found in chloroform
fraction followed by petroleum ether fraction. Both the fractions were subjected to column
chromatography for the isolation of secondary metabolites. The chloroform fraction (20.0g)
was chromatographed on a silica-gel column (550g, 60–120 mesh),eluted with petroleum
ether (100%) followed by gradient mixtures of petroleum ether−ethyl acetate in the
increasing order of polarity (9.5:0.5 → 3.0:7.0). In all 394 fractions of 100 mL each were
collected. On the basis of similar Rf values, color, and shape of spots in definite
circumstances, all the fractions were pooled together into eight fractions coded as A–H.
These fractions were further purified by column chromatography and crystallization. Fraction
B was crystallized from petroleum ether-ethyl acetate to yield the yellow solid compound
which on the spectroscopic examination and comparison of the data reported in the literature
identified as biochanin A (CMP-4). Fraction C was subjected to silica gel CC (petroleum
ether-ethyl acetate, 8.5:1.5→7.0:3.0) to give subfractions C-1 to C-6. Subfractions 3-5 were
crystallized from petroleum ether-ethyl acetate to afford yellow solid compound identified as
genisten (CMP-5).Similarly fraction D was subjected to column chromatography (petroleum
ether-ethyl acetate, 7.5:1.5→4.0:6.0) to give subfraction D-1 to D-15.On the basis of TLC D4 to D-12 were pooled together, evaporated to dryness in a rotary evaporator under reduced
pressure at 35oC to afford a yellow solid compound which after washing with methanol,
2
identified as pratensein (CMP-6). The fraction E was subjected to column chromatography
using petroleum ether-ethyl acetate (6.3:3.7) as the eluent. The major component on
crystallization from DCM yielded yellow solid. The compound after spectroscopic analysis
and comparison with literature data was identified as chrysoeriol (CMP-7). Fraction H was
subjected to silica gel CC (DCM: MeOH 9.3:0.7→8.5:1.5) to give subfractions H-1 to H-19.
Subfractions 4-16 were crystallized from DCM-MeOH to afford yellow solid compound
identified as luteolin (CMP-8). The hexane fraction (10.0g) was chromatographed on a silicagel column (300g, 60-120 mesh), eluted with petroleum ether (100%) followed by gradient
mixtures of petroleum ether-ethyl acetate in the increasing order of polarity (9.5:0.5 →
4.0:6.0). In all 195 fractions of 100 mL each were collected. On the basis of similar Rf values
of spots, all the fractions were pooled into four fractions coded as AH–DH. The fraction BH
was subjected to CC using petroleum ether-ethyl acetate (9.5:0.5 → 7.0:3.0) as the eluent to
give subfraction B-1 to B-37. On the basis of TLC screening, subfractions BH-1 to BH-12,
BH-15 to BH-24,BH-26 to BH-35 were pooled respectively and evaporated to dryness in a
rotary evaporator under reduced pressure at 35oC to afford white solid compounds which on
structural elucidation named as stigmasterol (CMP-1), oleanolic acid-3-acetate (CMP-2),
oleanolic acid (CMP-3) respectively.
2.4 Spectroscopic analysis for the characterisation of isolated compounds
The structure elucidations of the isolated compounds were carried out by various
spectroscopic techniques (1H NMR, 13C NMR, DEPT, IR and MS) .The chemical structures
of all these characterized compounds are depicted in Fig. 1. The spectral data of all the
characterized compounds were found in agreement with already reported in the literature [4].
The spectra for all these compounds are provided as a supplementary data (supplement 1).
3
1. CMP-1 (1H NMR)
1. CMP-1 (13C- NMR)
1. CMP-1 (MS)
4
2. CMP-2 (1H NMR)
2. CMP-2 (13C NMR)
2. CMP-2 (MS)
5
3. CMP-3 (1H NMR)
3. CMP-3 (MS)
6
4. CMP-4 (1H NMR)
4. CMP-4 (13C NMR)
4. CMP-4 (MS)
7
5. CMP-5 (1H NMR)
5. CMP-5 (13C NMR)
5. CMP-5 (MS)
8
6. CMP-6 (1H NMR)
6. CMP-6 (13C NMR)
6. CMP-6 (MS)
9
7. CMP-7 (1H NMR)
7. CMP-7 (13C NMR)
7. CMP-7 (MS)
10
8. CMP-8 (1H NMR)
8. CMP-8 (13C NMR)
8. CMP-8 (MS)
11
Figures
Figure S1: Calibration curves of standard solutions CMP 1-8 of different concentrations
100–800 ng per band.
12
Tables
Table S-1. Peak purity test for markers CMP 1-8
Markers
r (s.m)a
Standard track
CMP-1(1)
CMP-2(2)
CMP-3(3)
CMP-4(4)
CMP-5(5)
CMP-6(6)
CMP-7(7)
CMP-8(8)
0.9996
0.9997
0.9999
0.9998
0.9995
0.9999
0.9996
0.9999
Sample track
0.9994
0.9999
1.0000
0.9997
0.9996
1.0000
0.9999
1.0000
r (m.e)b
Standard track
0.9996
0.9998
1.0000
0.9996
0.9999
1.0000
0.9998
0.9996
Sample track
0.9993
0.9996
0.9999
0.9998
0.9993
0.9999
0.9999
0.9999
a) Correlation of spectrum at start of peak with spectrum at the centre of peak.
b) Correlation of spectrum at center of peak with spectrum at the end of peak.
Table S-2. Recovery study of markers CMP 1-8 by HPTLC.
Marker compounds
Amount present in
the sample (ng)
Amount
added (ng)
Amount
found(ng)
Recoverya
(%)
Mean
%
RSD%b
405.21
310.22
340.55
100
120
150
503.60
429.18
438.18
98.39
99.13
97.63
98.38
0.89
380.41
352.49
347.62
100
120
150
479.82
471.80
495.11
99.41
99.42
98.32
99.05
0.73
220.48
282.14
251.58
100
120
150
318.12
380.68
400.13
97.64
98.54
99.03
98.40
0.69
160.25
170.75
175.25
100
120
150
259.71
290.31
324.73
99.46
99.63
99.65
99.57
0.99
421.72
432.63
412.25
100
120
150
520.81
551.23
561.75
99.09
98.00
99.66
98.90
0.71
321.25
325.25
310.10
100
120
150
420.41
444.62
459.21
99.16
99.47
99.40
99.34
0.96
200.20
220.25
218.69
100
120
150
299.35
339.67
367.23
99.15
99.51
99.02
99.53
0.86
99.47
98.77
99.60
99.28
0.79
CMP-1
CMP-2
CMP-3
CMP-4
CMP-5
CMP-6
CMP-7
CMP-8
520.25
100
609.72
511.72
120
630.25
529.27
150
678.67
a) Recovery (%) = (amount found − original amount)/amount spiked × 100%.
b) RSD (%) = (SD/mean) × 100%.
13
Table S-3. Robustness testing of HPTLC densitometric method for markers CMP 1-8 (n= 6)
Parameters
CMP-1
SD/%RSD
CMP-2
SD/%RSD
CMP-3
SD/%RSD
CMP-4
SD/%RSD
CMP-5
SD/%RSD
CMP-6
SD/%RSD
CMP-7
SD/%RSD
CMP-8
SD/%RSD
Mobile phase
composition
1.21/0.91
1.0 9/0.82
1.62/0.89
1.51/0.99
1.51/1.00
1.59/1.38
1.39/1.09
1.53/1.43
Amount of mobile phase
2.58/1.31
1.59/1.21
1.81/1.43
1.31/1.01
1.75/1.04
2.07/1.01
1.09/0.53
2.42/1.29
Temperature
1.39/0.99
1.09/0.47
1.47/0.83
1.27/0.92
1.91/0.94
1.81/0.89
1.72/0.97
1.99/0.83
Relative humidity
2.03/0.99
1.29/0.82
1.33/0.99
1.48/1.00
1.47/0.98
2.06/1.04
2.03/0.98
1.71/0.98
Plate treatment
0.70/0.52
0.92/0.45
0.98/0.45
1.07/0.65
1.09/1.03
1.80/0.75
1.07/0.73
2.01/1.19
Time for spotting to plate
development
Time from development
1.52/0.77
0.98/0.47
1.21/0.77
2.01/1.04
1.05/0.46
2.02/1.09
2.05/0.85
1.09/0.67
1.53/0.63
1.18/0.87
1.01/0.79
1.61/0.43
1.98/0.42
1.47/0.97
1.46/0.68
2.01/0.51
to scanning
SD = Standard deviation of areas. %RSD = Relative Standard Deviation (consist of the average of three
concentrations 100,200 and 300ng/band for each component
Table S-4. Cytotoxic profile of isolated compounds using MTT assay.
S.No
markers
Concentration
(ug/ml)
1.
CMP-3
2.
CMP-4
3.
CMP-5
4.
CMP-6
5.
CMP-7
6.
CMP-8
1
10
25
50
100
1
10
25
50
100
1
10
25
50
100
1
10
25
50
100
1
10
25
50
100
1
10
25
50
100
% Cytotoxicity
B16-F10
4±5.7
30±2.5
46±7.3
56±2.6
64±4.3
4.5±6.7
33±7.4
58±2.5
88±1.3
90±0.6
12±3.2
28±1
32±1.5
80±1
89±0.8
3±2
11±4.7
26±2.7
67±1
74±7
5±1.5
41±2
60±2.5
75±1.8
78±1.6
24±2
68±0.5
74±1.5
89±0.7
90±0.5
IC50
30
18.4
41.6
35
17
3.5
A-431
2±1
7±1.2
10±1
22±2.5
32±1.9
9±2.8
18±2.5
42±6
88±0.2
89±0.3
8±4.6
16±0.3
22±1
53±4
87±0.8
12±6
31±2
41±4.4
50±2.4
63±2.8
17±3.5
46±3.5
55±0.6
63±1.8
73±1.4
13±4.2
34±3.6
50±3.4
67±2.5
74±4.2
IC50
18.1
34.0
60.3
46.1
15.4
25.6
HaCaT
4
8
11
30
78
82
14
47
59
23
57
60
49
53
56
47
66
73
-
14
References
[1]
de Almeida, J. G. L., Silveira, E. R., Pessoa, O. D. L., Magn. Reson. Chem. 2008, 46,
103-106.
[2]
Owen, R., Haubner, R., Mier, W., Giacosa, A., et al., Food Chem. Toxicol. 2003, 41,
703-717.
[3]
Hwang, J. T., Oh, H.-M., Kim, M.-H., Jeong, H. J., et al., Molecules 2014, 19, 1030910319.
[4]
Borges, C., Martinho, P., Martins, A., Rauter, A., Ferreira, M., Rapid Commun. Mass
Spectrom. 2001, 15, 1760-1767.
15