Industrial Crops and Products 76 (2015) 11331141

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Quantication of multianalyte by UPLCQqQLIT MS/MS and in-vitro

anti-proliferative screening in Cassia species
Preeti Chandra a,b , Renu Pandey a,b , Brijesh Kumar a,b, , Mukesh Srivastva b,c ,
Praveen Pandey d , Jayanta Sarkar d , Bhim Pratap Singh e
a

Sophisticated Analytical Instrument Facility, CSIRCentral Drug Research Institute, Lucknow 226031, Uttar Pradesh, India
Academy of Scientic and Innovative Research (AcSIR), New Delhi 110025, India
c
Biometry and Statistics Division, CSIRCentral Drug Research Institute, Lucknow 226031, Uttar Pradesh, India
d
Biochemistry Division, CSIRCentral Drug Research Institute, Lucknow 226031, Uttar Pradesh, India
e
Department of Biotechnology, Mizoram University, Tanharil, Aizawl, Mizoram 796004, India
b

a r t i c l e

i n f o

Article history:
Received 15 April 2015
Received in revised form 10 August 2015
Accepted 11 August 2015
Keywords:
Cassia species
UPLC-QqQLIT MS/MS
Quantication
In-vitro anti-proliferative activity
Principal component analysis

a b s t r a c t
Genus Cassia is widespread medicinal plants used in traditional system of medicines for the treatment of
various ailments. A new, rapid and sensitive method has been developed using ultra performance liquid
chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometry in negative electrospray ionization to determine content and distribution of eighteen bioactive compounds in different
plant parts of ve Cassia species. Multiple reaction monitoring was used for quantitative analysis. The
calibration curve obtained good linear regression relationship (r2 , 0.99860.9999) over the wide concentration range of all analyte. The recoveries ranged from 97.75% to 105.09 % (RSD 2.42%). Relative
standard deviations of intra-day and inter-day precisions were less than 3.37%. The validated method
was successfully applied to investigate chemical variations of anthraquinones, phenolics, avonoids and
terpenoids in leaf, stem, seed/pulp, ower and root. Principal component analysis depicted remarkable
chemical variation of bioactive compounds among Cassia species. The in-vitro anti-proliferative screening of methanol extracts were evaluated by SRB assay against the DLD1, A549, MCF-7, DU145 and FaDu
cell lines which showed >50% growth inhibitory effect. The present comprehensive evaluation is of great
importance for the application and quality control of studied Cassia species.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Indian medicinal plants are commonly used in home remedies against multiple ailments due to their pharmacologically
active principles and compounds (Gupta, 2010). Cassia is a
indigenous plant in Africa, Latin America, Northern Australia
and Southeast Asia. Several Cassia species are of high commercial and medicinal signicance since they are used as spices
and in traditional medicines (Danish et al., 2011). The most
widely recognized species of this genus are Cassia auriculata,
Cassia stula, and Cassia occidentalis followed by Cassia siamea
and Cassia uniora. Cassia species are reported to have various
pharmacological activities such as anti-bacterial (Wadekar et al.,

Corresponding author at: Sophisticated Analytical Instrument Facility,

CSIRCentral Drug Research Institute, Lucknow 226031, Uttar Pradesh, India.
Fax: +91 0522 2623405.
E-mail address: brijesh kumar@cdri.res.in (B. Kumar).
http://dx.doi.org/10.1016/j.indcrop.2015.08.030
0926-6690/ 2015 Elsevier B.V. All rights reserved.

2011), analgesic, anti-inammatory and anti-arthritic (Chaudhari

et al., 2012; Manogaran and Sulochana, 2004; Nsonde Ntandou
et al., 2010), hepatoprotective (Chauhan et al., 2009), anti-tumor
(Gupta et al., 2000), anti-fertility (Yadav and Jain, 2009), antifungal (Singh and Karnwal, 2006), anti-oxidant (Danish et al.,
2011; Siddhuraju et al., 2002), anti-leishmaniatic (Sartorelli
et al., 2007), anti-microbial (Ali et al., 1999), CNS (Mazumder
et al., 1998) and hypoglycaemic activity (Bhakta et al., 2001;
Chauhan et al., 2009; Nirmala et al., 2008; Pari and Latha,
2002).
The taxonomy and nomenclature of Cassia species are quite
complex. It is very difcult to differentiate them due to their overlapping morphological characters and close similarities (Kumar
et al., 2007). This usually leads to misidentication and misinterpretation of the components. Different class of compounds reported
from Cassia species are anthraquinones, phenolics, avonoids,
chromenes, terpenes, proanthocyanidins, coumarins, chromones,
aromatic compounds and lignans (Danish et al., 2011; Lee et al.,
2001 Zhao et al., 2013).

1134

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

Fig. 1. UPLCMRM extracted ion chromatogram of analytes (a) protocatechuic acid, (b) chlorogenic acid, (c) epicatechin, (d) caffeic acid, (e) catechin, (f) rutin, (g) ferulic
acid, (h) naringin, (i) quercetin, (j) luteolin, (k) apigenin, (l) kaempferol, (m) rhein, (n) emodin, (o) chrysophanic acid, (p) physcion, (q) oleanolic, (r) ursolic acid and internal
standard i.e., (s) curcumin.

Fig. 2. Graphical representation of content (g/g) of 18 compounds in ve Cassia species.

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

Till now, a series of methods, such as high performance thinlayer chromatography (HPTLC) (Bhope et al., 2010; Kadarkarai,
2011; Shailajan et al., 2013), multi-wavelength high-performance
liquid chromatography (Ni et al., 2009), high-performance liquid
chromatography (HPLC) (Chewchinda et al., 2014; Lai et al., 2010;
Mehta, 2012; Ni et al., 2009; Prakash et al., 2007; Sakulpanich
et al., 2012), gas chromatography/mass spectrometry (GCMS)
(Tzakou et al., 2007), have been reported for the determination of
anthraquinones, proanthocyanidins and/or phenolics.
However, these methods suffer from low sensitivity, low
resolution and/or long analysis time with large amount of solvents consumption (Chewchinda et al., 2014; Ni et al., 2009;
Prakash et al., 2007). Due to above discussed limitations; these
reported methods are being increasingly superseded by ultraperformance liquid chromatographytandem mass spectrometry
(UPLCMS/MS). UPLC-triple quadrupole/linear ion trap (QqQLIT )
mass spectrometer needs very shorter run time and low consumption of solvents. It enables rapid detection of the selected analytes
at very low concentration levels due to its selectivity and superior
sensitivity. As a preferred technique multiple reaction-monitoring
(MRM) mode serves for sensitive, accurate and simultaneous quantitation of selected analytes in complex matrices.
Genus Cassia is in great demand due to its medicinal properties as newer formulations are continually appearing in the market.
The quality and safety of the products has to be checked regarding
adulteration with misidentied species, which result in variations
in phytoconstituents. Nevertheless, to the best of our knowledge,
there is no report on the use of UPLCQqQLIT technique for analyzing targeted compounds in different plant parts of Cassia species. It
is therefore important to develop an efcient method, which will
allow the discrimination in terms of distribution of bioactive compounds in different plants parts of Cassia species for quality control
purposes.
In this study, we have attempted to develop a method based
on UPLCQqQLIT tandem mass spectrometric technique employing an MRM scan approach for quantifying multi-components in
negative ionization mode, which allows the simultaneous determination of anthraquinones, phenolics, avonoids, terpenoids, and
their comprehensive evaluation in different plants parts (leaf, stem,
seed/pulp, ower and root) of ve Cassia species viz., C. auriculata,
C. stula, C. occidentalis, C. siamea and C. uniora. Principal component analysis (PCA) was applied to study chemical variations of

1135

bioactive compounds among Cassia species. Further, the samples

were screened to study their in-vitro anti-proliferative activity.
2. Experimental
2.1. Reagents, chemicals and materials
Acetonitrile, Methanol (LCMS grade) and formic acid (analytical grade) were purchased from Fluka, SigmaAldrich (St. Louis,
MO, USA) and ultra high purity water was prepared using a MilliQ water purication system (Millipore Corporation, Bed-ford, MA).
Syringe lters (0.22 m) were purchased from Millipore (Billerica,
MA, USA). The reference standards (purity 96%) of chrysophanic
acid, physcion, rhein, emodin, apigenin, kaempferol, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, catechin,
epicatechin, ursolic acid, oleanolic acid and curcumin were purchased from SigmaAldrich Ltd. (St. Louis, MO, USA). Analytical
standards of (purity 95%) quercetin, naringin, rutin and luteolin
were purchased from Extrasyntheses (Genay, France). DMEM (Dulbeccos Modied Eagles Medium) and FBS (Foetal Bovine Serum)
were purchased from SigmaAldrich Ltd. (St. Louis, MO, USA).
Plant materials (leaf, stem, root, seed and ower of C. auriculata, C. occidentalis, C. siamea, C. uniora, with fruit pulp instead of
ower in case of C. stula) were collected from the plants grown in
western ghats of Maharashtra region between the months of May
to September, 2013. Voucher specimens of C. auriculata (ARC-5),
C. stula (ARC-2), C. occidentalis (ARC-3), C. siamea (ARC-1) and C.
uniora (ARC-4) were deposited in the Botanical Survey of India,
Western Region Centre, Pune, India.
2.2. Extraction and sample preparation
The dry plant parts of selected Cassia species were powdered
to a homogeneous size by a pulverizer and sieved through a 40mesh sieve, respectively. The dried powder of each part (5 g) was
weighed precisely and sonicated with 200 mL of 100% methanol for
30 min at room temperature using ultrasonic water bath (53 KHz)
and left for 24 h at room temperature. Three replicates of the
extraction process were carried out on each individual sample. The
solution was ltered through Whatman lter paper and evaporated to dryness under reduced pressure using rotatory evaporator
(Buchi Rotavapor-R2, Flawil, Switzerland) at 40 C. Dried residues

Table 1
Validation parameters for 18 reference analytes.
Analytes

Protocatechuic acid
Chlorogenic acid
Epicatechin
Caffeic acid
Catechin
Rutin
Ferulic acid
Naringin
Quercetin
Luteolin
Apigenin
Kaempferol
Rhein
Emodin
Chrysophanic acid
Physcion
Oleanolic acid
Ursolic acid
a

Regression equation

y = 8.32x 0.04
y = 18.2x + 0.01
y = 2.69x 0.02
y = 12.68x + 0.09
y = 37.80x + 0.11
y = 3.64x 0.01
y = 9.75x 0.01
y = 0.73x 0.001
y = 13.43x 0.05
y = 0.02x 0.01
y = 1.13x + 0.22
y = 0.01x 0.003
y = 0.62x 0.09
y = 34.00x 1.3
y = 28.81x + 0.6
y = 252.91x + 3518.2
y = 1.07x + 2.2
y = 0.03x + 5.65

Correlation coefcient.

r2 a

0.9993
0.9999
0.9991
0.9995
0.9998
0.9995
0.9991
0.9993
0.9992
0.9998
0.9998
0.9999
0.9986
0.9997
0.9990
0.9995
0.9997
0.9998

Linear range
(ng/mL)

150
150
1100
150
10250
150
5100
10250
150
0.550
0.5100
0.5100
5100
1100
1100
101000
1100
0.5100

LOD (ng/mL)

0.12
0.16
0.19
0.08
2.12
0.18
1.10
1.34
0.18
0.06
0.04
0.02
0.66
0.20
0.15
2.12
0.02
0.04

LOQ (ng/mL)

0.36
0.48
0.56
0.28
2.40
0.52
1.43
3.88
0.52
0.31
0.29
0.27
1.95
0.55
0.49
2.38
0.57
0.22

Precision RSD (%)

Stability

Intra-day
(n = 6)

Inter-day
(n = 6)

RSD
(n = 5)

1.66
0.86
2.22
1.72
1.24
1.13
2.51
0.63
1.02
0.77
3.37
0.88
1.52
0.62
1.58
0.62
0.38
0.44

1.05
2.63
2.94
1.82
1.36
1.14
1.23
2.36
1.17
2.25
0.93
0.91
4.09
1.77
2.10
1.36
1.38
1.27

2.78
1.66
1.68
2.11
2.15
3.19
2.86
1.89
1.66
1.59
2.68
2.45
2.16
0.39
1.20
1.29
0.64
0.72

Recovery RSD (%)

1.22
1.56
0.55
1.27
2.42
1.66
0.86
0.78
1.45
2.38
1.89
1.87
1.59
0.63
2.11
1.92
0.58
1.36

1136

Table 2
Contents of the 18 reference analytes in Cassia species (g/g).
Protocatechuic
acid

Chlorogenic
acid

Epicatechin

Caffeic
acid

Catechin Rutin

Ferulic
acid

Naringin Quercetin Luteolin

Apigenin Kaempferol

Rhein

Emodin

Chrysophanic
acid

Physcion Oleanolic Ursolic

acid
acid

C. siamea LF
C. siamea ST
C. siamea RT
C. siamea SD
C. siamea FL
C. stula LF
C. stula ST
C. stula RT
C. stula SD
C. stula PL
C. occidentalis LF
C. occidentalis ST
C. occidentalis RT
C. occidentalis SD
C. occidentalis FL
C. auriculata LF
C. auriculata ST
C. auriculata RT
C. auriculata SD
C. auriculata FL
C. uniora LF
C. uniora ST
C. uniora RT
C. uniora SD
C. uniora FL

13.4
33.62
1125
bdl
4.88
22.1
307.6
41.7
38.0
32.1
7.00
bdl
114
nd
nd
nd
nd
36.4
nd
bdl
1215
570
159
4020
4215

3035.2
4835
125.8
213.16
1495
180
248
193
178.46
187
189.52
191.5
197.5
197.5
187
220.23
197.5
180.5
198.5
88.2
213.5
76.8
183.5
86.6
83.2

24.4
nd
232
nd
nd
nd
12350.32
nd
nd
2360
nd
nd
bdl
nd
nd
54.2
26400
1638
36.4
119.2
398
nd
21.75
35.4
151.2

7350
555
1670.26
156.5
477.5
322.5
1200
149
52.5
145
223.5
289.5
177.3
149.5
110
156.5
204.5
130
174.5
305.5
92.5
201.5
194.5
645
112.5

bdl
nd
20.61
2.66
10.28
1356.23
1662.65
1785.13
bdl
1668
bdl
16.6
bdl
24.3
bdl
25.6
2440
1002
30.8
66.2
332
1.13
34.2
60.6
104

7.50
55.5
37.35
nd
7.75
10.44
127.16
nd
nd
nd
63.5
86.14
14.55
bdl
bdl
6.66
36.4.25
3.06
42.0
nd
55.4
92.8
172.8
236
137.2

nd
nd
nd
nd
nd
61.20
nd
nd
nd
nd
120.81
1.56
nd
nd
nd
nd
bdl
bdl
bdl
bdl
nd
nd
nd
nd
nd

1520
nd
nd
bdl
nd
nd
bdl
bdl
bdl
nd
nd
71.7
334
nd
bdl
bdl
nd
nd
bdl
bdl
bdl
bdl
nd
nd
nd

1360.43
nd
nd
nd
nd
995.2
7890
866
bdl
14600
nd
nd
bdl
bdl
bdl
bdl
bdl
nd
nd
nd
bdl
bdl
nd
nd
nd

117
195.23
2755
84.5
121
87.51
156.5
98.5
93.0
84.13
93.52
145
560
126.5
84.5
89.5
106
164.5
233
98.0
122.5
132.0
213.5
745.0
125.5

137.4
592.12
13660
17.40
22.6
79.15
114.8
164.4
nd
245.33
bdl
86.0
612
nd
nd
nd
67.6
484
15.74
nd
nd
21.4
950
nd
34.1

880.11
2925.22
5950
bdl
1850.55

179.5
32.0
bdl
43.35
58.5
95.6
1030
303.5
nd
nd
35.6
276
19.65
nd
35.4
43.1
267.5
nd
2.52

625
645
830
250.13
244
2205
5350
690.16
665.24
895
590.34
262
250
610.24
bdl
16650.12
5550
635
4685.11
9000
45550
650
625.23
8000.25
9950

ndnot detected, bdlbelow detection limit, LFleaf, STstem, RTroot, SDseed, FLower, PLpulp.

bdl
246
3140
nd
1.46
32.64
67.8
bdl
nd
nd
nd
nd
65.0
bdl
bdl
402
354
57.0
11.48
508
5440.23
nd
9.24
1706
3640

1830
2115.41
1440.45
925
2050
950
1785
1010
935
930
1100
1325
4385
1245
935
1160.35
1095
930
1050
2005
9050
1040
1215.3
9250
12200

nd
nd
827
nd
961
nd
nd
nd
nd
3280
nd
nd
nd
nd
66.2
nd
nd
bdl
bdl
bdl
bdl
nd
nd
nd
nd

110.25
3500
105600
nd
nd
nd
69.4
14.74
nd
bdl
nd
nd
310
nd
nd
nd
119.4
478
nd
nd
7.08
nd
172.6
nd
24.2

66.0
10300
39450.16
nd
17.78
nd
334
37.2
nd
nd
nd
nd
776
nd
nd
nd
169.2
1044
nd
nd
24.2
nd
446.31
52.5
6.32

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

Samples

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

1137

Fig. 3. (a) PCA score plot of 18 compounds distributed in different Cassia species. (b) PCA loading plot of different plants parts (leaves, stem, root, ower, seed/pulp) of Cassia
species.

(5 mg) were used for in-vitro anti-proliferative activity screening and (1 mg) were weighed accurately and dissolved in 1 mL
of methanol using ultrasonicator (Bandelin SONOREX, Berlin). The
solutions were ltered through 0.22 m syringe lter (Millex-GV,
PVDF, Merck Millipore, and Darmstadt, Germany). The ltrates
were diluted with methanol to nal working concentration. 50 L
of internal standard (IS) were spiked in nal working solution, vortexed for 30 s and 5 L aliquot was injected into the UPLCMS/MS
system for analysis.

2.3. Preparation of standard solution

A mixed standard stock solution containing anthraquinones
(chrysophanic acid, physcion, rhein, emodin), phenolics (caffeic
acid, ferulic acid, protocatechuic acid, chlorogenic acid), avonoids
(quercetin, kaempferol, apigenin, naringin, catechin, rutin, epicatechin and luteolin), triterpenoid (ursolic acid, oleanolic acid)
was prepared in methanol. Mixed standards were diluted with
methanol within the ranges from 0.5 to 250 ng/mL to prepare

1138

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

working standard solutions which was used for plotting calibration curve. The internal standard, curcumin for negative mode was
spiked to each concentration solution at a nal concentration of
50 ng/mL (50 L of internal standards mixture of 1000 ng/mL of curcumin in methanol) were mixed properly. The standard stock and
working solutions were all stored at 20 C until use and vortexed
prior to injection.

2.4. Instrumentation and analytical conditions

The system used was an Acquity ultra-performance liquid chromatography (UPLC) consisting of an autosampler and a binary
pump (Waters, Milford, MA) equipped with a 10 L loop (partial
loop injection mode). Compounds were separated using an Acquity
BEH C18 (2.1 50 mm, 1.7 m; Waters, Milford, MA) analytical column at a column temperature of 30 C. The mobile phase consisted
of two solvents: 0.1% (v/v) formic acid water (A) and acetonitrile
(B) at a ow rate of 0.4 mL/min. The gradient program performed
of an initial linear increase from 5% to 90% B over 5 min, followed
by hold of 90% B over 1 min, then return to the initial condition over
2 min with a sample injection volume of 5 L.
The UPLC system was coupled to triple-quadrupole linear ion
trap mass spectrometer (API 4000 QTRAPTM MS/MS system from
AB Sciex, Concord, ON, Canada) equipped with electrospray (Turbo
VTM ) ion source was operated in negative ionization mode. The
optimized parameters for negative mode were as follows: the ion
spray voltage was set to 4200 V, the turbo spray temperature,
550 C; nebulizer gas (gas 1), 20 psi; heater gas (gas 2), 20 psi; collision gas, medium; the curtain gas (CUR) was kept at 20 psi.
Optimization of the mass spectrometric conditions was carried
out by infusing 50 ng/mL solutions of the analytes dissolved in
methanol at 10 L/min using a Harvard 22 syringe pump (Harvard Apparatus, South Natick, MA, USA). For the MRM quantitation,
highest abundance of precursor-to-product ions for each compound was chosen. The dwell time for both the parent and the
internal standard was set at 200 ms. For, full scan ESIMS analy-

sis, the spectra covered the range from m/z 100 to 1000. Analyst
1.5.1 software package (AB Sciex) was used for instrument control
and data acquisition.
2.5. Cell lines and anti-proliferative assay
Human cancer cell lines A549 (Lung carcinoma), MCF-7 (Breast
adenocarcinoma), DU 145 (Prostate carcinoma), DLD-1 (Colorectal adenocarcinoma), FaDu (squamous cell carcinoma of pharynx)
were obtained from American Type Culture Collection (ATCC), USA.
These cells were cultured in DMEM supplemented with 10% FBS and
antibiotic combinations in 5% CO2 humidied atmosphere at 37 C.
A colorimetric sulforhodamine B (SRB) assay is the mostly
preferred technique and was used for the measurement of antiproliferative activity (Adaramoye et al., 2011; Fricker and Buckley,
1996; Keepers et al., 1991; Skehan et al., 1990). This basically
depends on the incur of the negatively charged pink amino xanthine dye, sulphorhodamine B (SRB) through basic amino acids in
the cells. The dye released will give more acute color and more
absorbance, when the number of cells and amount of dye is taken
up is greater, after xing, the cells are lysed (Skehan et al., 1990).
The SRB assay is sensitive, reproducible and gives better linearity, a
good signal-to-noise ratio and has a stable end-point that does not
require a time-sensitive measurement (Fricker and Buckley, 1996;
Keepers et al., 1991). Ten thousand cells were seeded to each well
of 96-well plate, grown overnight and exposed to test samples at
100 g/mL concentration for 48 h. Cells were then xed with icecold trichloro acetic acid (50% w/v, 50 L/well), stained with SRB
(0.4% w/v in 1% acetic acid, 50 L/well), washed and air dried. Bound
dye was dissolved in 150 L of 10 mM Tris base and plates were read
at 510 nm absorbance (Epoch Microplate Reader, Biotek, USA).
Anti-proliferative activity of test samples was calculated as:
%inhibition in cell growth

100

Absorbance of compound treated cells

Absorbance of untreated cells


100

Table 3
Percentage (%) growth inhibition in various plant parts of Cassia species.
Percentage growth inhibition at 100 g/mL concentration
DLD1

FaDu

DU145

A549

MCF-7

C. auriculata-LF
C. auriculata-ST
C. auriculata-RT
C. auriculata-SD
C. auriculata-FL
C. stula-LF
C. stula-ST
C. stula-RT
C. stula-PL
C. stula-SD
C. occidentalis-LF
C. occidentalis-ST
C. occidentalis-RT
C. occidentalis-SD
C. occidentalis-FL
C. siamea-LF
C. siamea-ST
C. siamea-RT
C. siamea-SD
C. siamea-FL
C. uniora-LF
C. uniora-ST
C. uniora-RT
C. uniora-SD
C. uniora-FL

49.99
65.08
68.59
51.92
67.61
27.05
26.51
25.38
14.89
11.11
33.48
18.88
13.98
36.02
12.76
12.1
14.69
70.92
7.10
19.13
68.59
15.48
25.67
19.94
31.38

75.1
63.59
73.28
68.82
63.48
6.39
10.53
7.40
14.72
25.33
22.34
0.97
23.2
30.91
24.44
5.69
7.32
74.01
10.26
7.51
73.28
36.33
46.75
55.69
68.78

77.22
90.62
91.96
40.17
67.57
27.71
37.76
23.55
6.33
7.34
25.09
8.48
6.20
36.08
28.78
15.51
2.45
55.91
3.79
31.99
91.96
20.60
26.16
32.06
43.32

18.23
46.55
44.9
54.97
33.45
36.22
14.82
13.61
13.35
1.9
23.65
5.43
10.14
2.25
9.64
14.64
1.16
52.26
1.57
17.17
44.90
16.44
4.43
44.99
47.49

59.54
79.6
51.6
56.04
39.02
46.06
8.86
15.69
10.72
16.24
27.63
6.51
3.62
0.95
31.54
31.49
2.41
62.74
4.17
16.44
51.6
15.01
4.30
50.69
48.8

Doxorubicin

83.35

98.7

70.99

72.58

82.01

LFleaf, STstem, RTroot, SDseed, FLower, PLpulp.

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

2.6. Principal component analysis

PCA was carried out based on the contents of eighteen bioactive
compounds in seed and leaf of ve Cassia species, using STATISTICA 7.0 software. When the amount of investigated compounds
were found below the quantitation limit or not detected than these
values were considered to be zero.
3. Results and discussion
3.1. Optimization of chromatographic and MS/MS conditions
Complete separation of proximate analytes is certainly not
required for MS/MS detection. In this study, chrysophanic acid
and emodin are having same product ion, while catechin and epicatechin are having same precursor and product ion. To obtain
better resolution various compositions of solvents were tried to
get a suitable mobile phase. Due to stronger elution ability of acetonitrile over methanol it was selected for this method. Similarly
an Acquity UPLC BEH C18 (2.1 50 mm, 1.7 m; Waters, Milford,
MA) column which was more suitable for acidic mobile phase
with smoother baseline was selected as compared to other tested
columns. Formic acid was found more effective for ionization of
compounds detected in the negative ESI mode. After testing various concentrations (0.05%, 0.1%, 0.2% and 0.3%) of formic acid 0.1%
formic acid concentration was nally selected. A gradient elution
with 0.1% formic acid in water and acetonitrile at a ow rate of
0.4 mL/min with column temperature of 30 C was resulted in separation of the 18 compounds in less than 8 min chromatographic
run time.
All the MS parameters for 18 compounds i.e., precursor ion,
product ion, declustering potential (DP), entrance potential (EP),
collision energy (CE) and cell exit potential (CXP) were optimized in negative ESI mode, by ow injection analysis (FIA).
The chemical structures of 18 components were characterized
based on their retention behavior and MS information such
as quasimolecular ions [MH] , fragment ions [MHCOO] ,
[MHCOOCH3 ] , [MCOH2 O] compared to related standards
and literatures (Pandey et al., 2014; Wei et al., 2013; Xia et al., 2011;
Yu et al., 2009). MRM parameters were optimized to achieve the
most abundant, specic and stable MRM transition for each compound as shown in Table S1. MRM extracted ion chromatogram of
18 analytes are shown in Fig. 1.
3.2. Analytical method validation
The proposed UPLCMRM method for quantitative analysis was
validated according to the guidelines of international conference on
harmonization (ICH, Q2R1) by linearity, LOQs and LODs, precision,
solution stability, and recovery.
3.2.1. Linearity, LOD and LOQ
The stock solution was diluted with methanol to different working concentrations for the construction of calibration curves. The
linearity of calibration was performed by the analytes-to-IS peak
area ratios versus the nominal concentration and the calibration
curves were constructed with a weight (1/x2 ) factor by leastsquares linear regression. The LODs and LOQs were measured with
S/N of 3 and 10, respectively as criteria. The results are listed in
Table 1. All the calibration curves indicated good linearity with
correlation coefcients (r2 ) from 0.9986 to 0.9999 within the test
ranges. The LODs for each analyte varied from 0.02 to 1.34 ng/mL
and LOQs from 0.06 to 3.88 ng/mL and were much lower than those
obtained with previous HPLC methods (Chewchinda et al., 2014; Ni
et al., 2009; Prakash et al., 2007).

1139

3.2.2. Precision, stability and recovery

Precision was determined by relative standard deviation (RSD)
with intra-day and inter-day variations, for the determination of
precision of the developed method were evaluated by determination of eighteen analytes in six replicates on a single day and
by duplicating the experiments over three successive days. The
overall intra-day and inter-day precision was not more than 3.37%.
Stability of sample solutions stored at room temperature was evaluated by replicate injections at 0, 2, 4, 8, 12 and 24 h. The stability
RSD% value of eighteen analytes is 3.19%. To evaluate the accuracy,
recovery test was applied by spiking three different concentration
levels (high, middle and low) of the analytical standards into the
samples. Three replicates were performed at each level. The analytical method developed had good accuracy with overall recovery
in the range from 97.75% to 105.09% (RSD 2.42 %) for all analytes
(Table 1).

3.3. Quantitative analysis of Cassia species

The proposed UPLCESIMS/MS method was applied to quantify 18 bioactive compounds in different plant parts of ve Cassia
species viz., C. auriculata, C. stula, C. occidentalis, C. siamea and C.
uniora. Remarkable differences in the quantity of anthraquinones,
phenolics, avonoids and terpenoids were observed during study
of quantitative result. In respect to the content of each analyte,
chrysophanic acid, physcion, ursolic acid, oleanolic acid, rutin and
luteolin are the most abundant bioactive compounds in majority of
parts of Cassia species as listed in Table 2. Anthraquinone like rhein
(14,600 g/g) was present in highest amount in pulp of C. stula,
while found below detection limit in other species, which clearly
showed the characteristic variation among ve Cassia species. The
results were also found consistent according to the previous reports
of determination of rhein (Chewchinda et al., 2014; Ni et al., 2009).
Other anthraquinones like emodin (2755 g/g), chrysophanic acid
(136,600 g/g) and physcion (5950 g/g) are considered as the
characteristic components as they were detected highest in C.
siamea root. In the similar pattern for terpenoids, oleanolic acid
(10,560 g/g) and ursolic acid (39,450.16 g/g) was detected highest in the root of C. siamea while found comparatively low and/or
not detected in other species, which shows characteristic pattern
and signicant variation among ve Cassia species. This clearly indicated that C. siamea root may be used as substitute for C. auriculata
and C. stula in various herbal formulations/preparations.
Higher content of avonoids such as rutin (45,550 g/g) and
quercetin (5440.23 g/g) were detected in the leaves, and luteolin (12,200.0 g/g) in owers of C. uniora with comparative lower
content in C. occidentalis and C. stula. But in contrast, kaempferol
(3280 g/g) was detected highest in pulp of C. stula. Phenolics such
as protocatechuic acid (4215 g/g) and ferulic acid (236 g/g) were
also found highest in ower and seed of C. uniora respectively,
while not detected in other few parts of Cassia species. Epicatechin (12,350.32 g/g) was detected in signicant highest amount
in stem of C. stula, which is in agreement to the reported literature
(Patidar et al., 2012). Graphical representations of these observations were shown in Fig. 2, which clearly explains the chemical
variations among ve Cassia species and their plant parts.
Contents of the bioactive compounds are directly responsible for
the efcacy hence change in their concentration may lead to the
variation. Therefore, it is important to select as many as possible
pharmacologically active chemical markers in order to distinguish
ve Cassia species with plant parts of different quantities. The quantitative results also indicated the importance of plant parts and
their impact on product quality. The overall quantitative analysis
indicated that this method has signicant importance in comprehensive evaluation of selected 18 compounds, which could be used

1140

P. Chandra et al. / Industrial Crops and Products 76 (2015) 11331141

as the markers for quality control of Cassia species and its related
preparations.
3.4. In- vitro anti-proliferative activity
Evaluation of in-vitro anti-proliferative activity by SRB assay
against the DLD1, A549, MCF-7, DU145 and FaDu cell line was done
with doxorubicin, which is used as positive control with percentage growth inhibition at 100 g/mL concentration. Doxorubicine
caused more than 70% growth inhibition in all the cell lines. Result
in Table 3 showed that root of C. siamea (74.01%) and C. auriculata
(91.96%) and leaf of C. uniora (68.59% and 73.28%) were able to
inhibit growth in DLD1 and FaDu cell lines, respectively. Similarly,
leaf of C. uniora (91.96%), stem of C. auriculata (90.62%) extracts
showed >70% growth inhibitory effect against Du145 cell line. Seed
and stem of C. auriculata (54.97% and 79.60%) were able to inhibit
>50% growth in A549 and MCF-7 cell lines.
3.5. Chemical evaluation of Cassia species by principal
component analysis
Principal component analysis (PCA) is the most widely used
chemometrics method for multivariate data analysis (El Senousy
et al., 2014). PCA was carried out based on the quantitative data
to compare and evaluate the quality of species based on the characteristics of the contents of eighteen investigated compounds in
leaves, stem, and root of ve Cassia species.
The PCA was studied on the basis of total matrix data of 18 compounds collectively in all plant parts of ve species. The PCA shows
that the data matrix reduced to 5 PCs explaining 86% variation.
Jointly the rst two PCs explain 54.76% variation of data where the
18 compounds were distributed in 3 clusters in the loading biplot as
shown in Fig. 3(a). This explained similarity in the pattern of quantities among plant parts of various species. However, score plot by
PC1 vs PC2 as shown in Fig. 3(b) indicated distribution of investigated samples in four clusters. From the loading scatter plot, it
was observed that different variables shows different contributions
in discrimination of Cassia species. However quantitative analysis
showed that leaf, stem and root of C. siamea, with leaf and seed of
C. uniora species were represented the characteristic pattern. The
C. siamea root is placed in an extreme of the biplot indicating high
quantity levels of most of the compounds.
PCA was also performed on the basis of whole data in individual
plants parts viz., leaf, stem, root, ower, seed/pulp of ve Cassia
species and explained in Fig. S1(a)(e), respectively. Depending
upon the multidimensional pattern in the quantity of compounds,
all Cassia species were individually classied and discriminated.
Thus, the PCA study clearly showed the remarkable variations
among the various plant parts of Cassia species.
4. Conclusions
A new approach for simultaneous quantitative analysis of 18
bioactive compounds was developed by MRM acquisition mode
using an UPLCQqQLIT tandem mass spectrometer in different plant
parts of Cassia species. The contents observed in quantitative analysis revealed highest content of anthraquinones in seed/pulp of
C. stula and C. auriculata, in comparison to other parts and other
Cassia species. Hence, it clearly suggested the suitability of these
parts as raw material in the manufacturing of Cassia based products. The comparative analysis of content of detected compounds
in all plants parts will help consumers to select one of them according to their requirement. Nevertheless, this method is simple, rapid
and sensitive which give useful quantitative ngerprint, to be
used for grading bioactive compounds commercially. Plant samples
were screened for their in-vitro anti-proliferative activity, in which

results were widely varied and could contribute to their application to different therapeutic areas. Principal component analysis
showed that there were remarkable differences in the distribution
of the chemical markers in various plant parts of Cassia species.
As an overall result, this is the rst time that chemical differences
among these different plant parts have been observed systematically. This method has signicant importance in comprehensive
evaluation of selected 18 compounds, which could be used as the
markers for quality control of Cassia species and its related preparations.
Acknowledgements
The authors gratefully acknowledge SAIF, CSIR-CDRI, Lucknow
where the mass spectrometric studies were carried out. Preeti
Chandra is thankful to CSIR, New Delhi for senior research fellowship.
CDRI communication number is 9053.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.08.
030.
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