American Journal of Anal yt ical Chemistry, 2011, 2, 871-878
doi:10.4236/ajac.2011.28100 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Extraction, Identification and Antioxidative Properties of
the Flavonoid-Rich Fractions from Leaves and Flowers of
Cassia angustifolia
Abdul Qayoom Laghari1, Shahabuddin Memon1*, Aisha Nelofar 2, Abdul Hafeez Laghari1,2
1National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro, Pakistan
2Pakistan Council of Scientific and Industrial Research, Karachi, Pakistan
Received August 17, 2011; revised September 26, 2011; accepted October 9, 2011
Abstract
Flavonoids identification, total flavonoids content and antioxidant activity of the extracts from the leaves as
well as flowers of Cassia angustifolia were determined. Five different extraction techniques were used for
the extraction of flavonoids from leaves and flowers. Total flavonoid contents (TFC) in the extracts were
determined by UV-visible spectrophotometry. Individual flavonoids were identified and quantified by
high-performance liquid chromatography coupled with photodiode array detection and electrospray ioniza-
tion tandem mass spectrometry (HPLC-PDA-ESI-MS). It has been examined that aqueous ethanol (70%)
fractions of C. angustifolia flowers and leaves are highly rich in flavonoids and microwave extraction is the
best method for the extraction of individual flavanoid constituents (1 - 9) as well as total flavonoid contents.
It was also found that compound 8 was absent in the leaves and compounds 2 and 5 could not be identified.
The extracts of C. angustifolia flowers and leaves show strong antioxidant activity.
Keywords: Cassia angustifolia, Flavonoids, Antioxidant Activity, HPLC-ESI-MS, Microwave Extraction
1. Introduction
Flavonoids are the polyphenolic phytochemicals with in-
consistent phenolic structures; they consist of flavones,
flavanone, flavanols, flavonols and flavanonols that com-
prise a large group of secondary metabolites in plants
[1-4]. They are found in vegetables, fruits, flowers, grains,
barks, roots and stems [5]. Flavonoids have significant
biological activities, such as anti-microbial [6], antioxi-
dant [7], anti-cancer [8] and anti-inflammatory activity
[9]. Present study was carried out to investigate flavon-
oids in the Cassia angustifolia due to its significance in
curing different diseases.
Cassia angustifolia Vahl. (Caesalpiniaceae) com-
monly known as Sanna makkai, grows in hot arid areas
of Pakistan and India. It’s a reputed drug in traditional
medicine and frequently used in folk medicine as a pur-
gative for a long time [10]. Most of pharmacopoeias of
the world have also recorded this plant [11]. Due to its
cathartic properties it is valued as a medicine and is par-
ticularly useful in habitual constipation. It is employed in
the treatment of amoebic dysentery as an anthelmintic
and as a mild liver stimulant [12]. It is extensively used
as febrifuge in splenic enlargements, typhoid, cholera,
anemia [13], laxative, genotoxicity, and toxicity in Es-
cherichia coli [14]. Leaves and pods of C. angustifolia
are traditionally used as purgatives; the most important
purgative constituents are Sennosides A, B, C, D,
emodin, isorhamnetin and essential oil [15]. Leaves of C.
angustifolia are also used as a safe laxative and this plant
contributes considerably to commercial drugs and has
been investigated in several parts of the world for various
therapeutic preparations in different ways [16].
Due to enormous usage of C. angustifolia in curing
different diseases and the work reported in the literature
concerning systemic study of chemical composition en-
couraged to further explore its active constituents re-
sponsible for bioactivity. In present study extraction of
flavonoids from the leaves and flowers of C. angustifolia,
has been done by using five different extraction tech-
niques. Identification of flavonoids, total flavonoid con-
tents and antioxidant activity of the different extracts was
also determined.

A. Q. LAGHARI ET AL. 872
2. Experimental
2.1. General
Formic acid, sodium nitrite, aluminum chloride, sodium
hydroxide were purchased from Merck Schuchardt OHG
85662 (Hohenbrunn, Germany). Butylated hydroxyani-
sole (BHA) was procured from BDH laboratory reagents
(Poole England). Acetonitrile, methanol, ethanol and
petroleum ether (40 - 60 Bp) (HPLC grade) were pur-
chased from Lab-Scan Analytical Science (Bankok
10330 Thailand).
2.2. Plant Material
The leaves and flowers of C. angustifolia were collected
in March 2010 from Nangar parkar district Mithi at
Tharparkar of Sindh province, Pakistan. Plant has been
identified by Dr. Abdul Jabbar Pirzada, Assistant Pro-
fessor, Institute of Plant Sciences, University of Sindh,
Jamshoro/Pakistan.
2.3. Extraction of Flavonoids
Fatty constituents from the leaves or flowers (coarsely
powdered and shade dried) were extracted with petro-
leum ether by soxhlet extraction. About one and half
hour was taken to extract all fatty constituents and sol-
vent became colorless when left for further extraction.
Then flavonoids extraction from defatted plant material
was performed by a reported method [17] with slight
modification.
2.3.1. Mi crowave Ext raction
1 g of defatted flowers or leaves were taken in vessel
already containing 50 mL of aqueous ethanol (70%), then
the vessel was placed into microwave decomposition
system (Start E Microwave Extraction system, Mile stone,
Model-Act 38-Revol-03106) for microwave-assisted
extraction. The temperature for extraction was 50˚C and
time for extraction was 9 min. After this the sample was
filtered and volume was made 50 mL in a volumetric
flask with aqueous ethanol (70%) solvent.
2.3.2. So xh l et Extraction
1 g of defatted flowers or leaves were taken and shifted
into filter paper thimble. 160 mL of aqueous ethanol
(70%) were poured into round bottom flask (250 mL
capacity) followed by fitting it on soxhlet assembly on
water bath. Extraction time was 5 h, until the clear solu-
tion was observed in Soxhlet, where thimble was placed.
Solvent was then evaporated under vacuum and the ex-
tract was diluted with same solvent and volume was
made 50 mL in volumetric flask.
2.3.3. So n i cation Extra ction
1 g of defatted flowers or leaves were taken and shifted
into a conical flask (100 mL capacity) containing 50 mL
of aqueous ethanol (70%). It was sonicated in ultrasonic
bath (Supersonic X-3, Model DSD80A5QS) for one hour.
It was then filtered and volume was made up to 50 mL in
volumetric flask in the same solvent.
2.3.4. Marina t ed Ex traction
1 g of defatted flowers or leaves were taken and shifted
into conical flask (100 mL capacity) containing 50 mL of
aqueous ethanol (70%). It was left for 48 h at room tem-
perature and filtered followed by dilution to make the
volume up to 50 mL in a volumetric flask.
2.3.5. Reflux Cond ensation Extracti on
1 g of defatted flowers or leaves were taken and shifted
into round bottom flask (100 mL capacity) containing 50
mL aqueous ethanol (70%). After fixing a condenser, it
was left for reflux condensation on heating mantel
(Isopad LG2/ER 100 mL, model 913323, Tmax-450˚C)
for two and half hours. Extract was filtered and diluted
up to 50 mL.
2.4. Determination of Total Flavonoid Contents
Total flavonoid contents were determined by a reported
method [17] with slight modification. The reaction mix-
ture was comprised of 2 mL plant extracts, 0.6 mL so-
dium nitrite (5% w/v), 0.5 mL of aluminum chloride
(10% w/v), 3 mL of sodium hydroxide (4.3% w/v) and
distilled water to make volume 10 mL. In each step, 6
min. time period was given for shaking to complete reac-
tion. Afterward, the solution was allowed to stand for 15
min. before measuring the absorbance. Measurement of
absorbance at 500 nm was performed on a Specord 200
analticjena UV-vis spectrophotometer (Germany), with a
1 cm cell. Aqueous ethanol (70%) was used as reference,
rutin was used as a standard and results were calculated
as rutin equivalents (Rutin eq., mg/mL) of C. angustifo-
lia.
2.5. Chromatographic Analysis of Flavonoids
The analysis of flavonoids in samples was carried out by
using HPLC-ESI-MS/MS. The HPLC conditions ad-
justed were as follows:
The liquid chromatograph system was equipped with
the photo diode array detector (PDA) and a vacuum de-
gasser. Separations were made by using Hypersil Gold
C18 (250 mm × 4.6 mm, 5 μm) (Thermo electron corpo-

A. Q. LAGHARI ET AL. 873
ration USA) column and analytical data was evaluated
by using X-Caliber data processing system (2.0 SR2).
The mobile phase was composed of methanol-acetoni-
trile (7:3) (A) and 0.1%, v/v formic acid in water (B).
The flow rate was 1 mL/min. The gradient programming
was as follows; starting from concentration of A at 5%
for 5 minutes and then gradual increase from 5% to 30%
in 10 minutes. Isocratic step of 5 minutes and then
brought back to 5% in 5 min followed by 5 min for col-
umn equilibration. The eluent was monitored using the
PDA detector set at three different wavelengths 270, 320
and 360 nm.
2.6. Identification and Qualitative Analysis of
Constituents by LC-MS
Identification of the constituents was performed with a
Thermo Finnigan LCQ Advantage ion trap mass spec-
trometer (Vernon Hills, Illinois, USA) fitted with the
liquid chromatograph system coupled on-line with an
Electro Spray Ionization (ESI) source and X-Caliber
software was used for data acquisition. Analytical condi-
tions were: positive ion mass spectra of the column elute
were recorded in the range m/z 200 - 700. Capillary tem-
perature was kept at 200˚C and Voltage 4.5 kV. Sheath
gas flow rate was kept at 70 (arbitrary unit). The result-
ing total ion chromatogram (Figures 1(a)-(b)) is a plot of
time vs area showing the total response of each constitu-
ent on the basis of its molecular ion abundance. Identifi-
cation of individual flavonoids in extracts was carried
out with the help of LC-MS-ESI by comparing their
masses and λmax with the literature data [18-24].
2.7. DPPH Antioxidant Activity
DPPH scavenging ability of C. angustifolia flowers or
1
2
3
4 5
6
7
8
9
m/z 291
m/z 277
m/z 295
m/z 499
m/z 235
m/z 409
m/z 315
m/z 453
0 5 10 15 20 25
Time (min)
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
(a)

A. Q. LAGHARI ET AL. 874
9
3
2
1
4
5
6
7
m/z 291
m/z 277
m/z 295
m/z 499
m/z 235
m/z 409
m/z 453
m/z 315
0 5 10 15 20 25
Time (min)
100
50
\
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
100
50
0
(b)
Figure 1. HPLC-MS chromatogram of the compounds identified from flowers (a) and leaves (b).
leaves extracts was measured by their bleaching capabil-
ity toward purple DPPH methanol solution. This assay
was performed according to the reported method [25,26]
by mixing 3 ml of 0.1 mM DPPH in methanol and 1 ml
of sample solution in aqueous ethanol (70%). The ab-
sorbance was measured on Specord 200 analticjena
UV-vis spectrophotometer (Germany) at 517 nm. De-
creasing amplitude of signal at the selected wavelength
confirmed a high radical scavenging activity. In addition,
the antioxidant activity of BHA as standard reference
was assayed; methanol was used as blank and the meas-
urement of solutions without sample were used as the
control. The inhibition of DPPH radicals by the samples
was calculated as follows:
DPPH inhibition (%) = [(A – B)/A] ×100. (1)
where A is the absorbance without extract and B is the
absorbance with the extract.
IC50 values were calculated by considering the 50%
inhibition of DPPH free radical in 5 min by flowers or
leaves samples at minimum concentration and comparing
with same response shown by BHA.

A. Q. LAGHARI ET AL.875
3. Results and Discussion
3.1. Identification and Quantification of
Flavonoids
Identification of the individual flavonoids was performed
through HPLC-PDA-ESI/MS, by comparing their masses
and λmax with the literature data (Table 1) [18-24]. Epi-
catechin (1), (-)-(2S)-6-Methoxy-[2”,3”:7,8]-furanofla-
vanone (3), Kaempferol 3-O-sulphate-7-O-c-arabi- no-
pyranoside (4), Vidalenolone (6), (2S)-7,8,bis-3’,4’-
(2,2-dimethyl-chromano)-5-hydroxyflavanone (7), 3,7-
dihydroxy-4’,8-dimethoxyflavone (8) and 14-hydroxyar-
tonin E (9) were indentified, but compounds 2 and 5
could not be identified, because the data does not match
with the literature, may be these compounds are new and
first time reported. Chemical structures of the identified
compounds are given in Figure 2; it shows that from
seven identified compounds, 3 and 7 are flavanones, re-
maining are flavones except compound 6, which is not a
flavonoid but a phenolic metabolite, i.e. Vidalenolone.
All of these are free flavonoids except compound 4
which contains arabinopyranoside moiety.
Flowers of C. angustifolia contains all 1-9 compounds
in varying quantities, on the other hand compound 8 was
absent in leaves. Microwave and soxhlet extraction tech-
niques are the best techniques to extract all 1-9 com-
pounds from the flowers and leaves, with exception of
compound 8, which was not found may be it is absent in
the leaves. While the compound 6 and 8 could not be
extracted by other extraction techniques like sonication,
marination and reflux condensation. Flowers contain
greater amount of total flavonoids than the leaves.
Quantification was performed in relative % by taking
area of all constituents from HPLC-MS chromatogram
and taking it as denominator to calculate each constituent
by putting area of that content in numerator and multi-
plying by 100. It was found that the relative percentage
of individual flavonoids varies in different extraction
techniques (Table 2). Relative percentage composition
of the compounds were higher in microwave extraction
techniques with few exceptions, compound 6 was higher
in soxhlet; both in flowers and leaves, while compound 1
and 5 were higher in reflux condensation extraction, but
compound 5 was higher only in leaves.
3.2. Total Flavonoid Contents
Among all the other extraction methods the total flavon-
oid contents (Table 3) have been found higher in mi-
crowave extraction, i.e. 28.15 mg/g and 26.3 mg/g in the
flowers and leaves, respectively. While lowest amount of
total flavonoid contents were found in case of flowers
and leaves extracts obtained by reflux condensation and
sonication extraction methods, respectively. As a result,
it could be suggested that microwave extraction tech-
nique is best option to extract flavonoids in higher
amounts.
3.3. Advantages and Disadvantages of the
Extraction Methods
Five different extraction techniques were used and ex-
traction efficiency was found to be dependent on all the
techniques. The purpose of using five different tech-
niques is to find out a convenient extraction method in
order to obtain more number and greater quantity of fla-
vonoids. However, microwave extraction method has
been found as the best option for the extraction of fla-
vonoids; it’s very easy, robust and least time consuming
Table 1. Flavonoid glycosides identified from the extracts of leaves and flowers by HPLC-PDA-ESI/MS.
Found
in samples
S. No. Identity λmax (nm)
Reported
λmax (nm)
Samples
Calculated
molecular
mass M + 1 MS2
1 Epicatechin 225, 267 224, 269 290 291 273
2 unknown - 232, 264 276 277 -
3 (-)-(2S)-6-Methoxy-[2”,3”:7,8]-furanoflavanone 234, 247, 341 233, 253, 338294 295 190
4 Kaempferol 3-O-sulphate-7-O-c-arabinopyranoside 270, 350 272, 345 498 499 287
5 unknown - 230, 274, 320498 499 -
6 Vidalenolone 224, 266 225, 267 234 235 203
7 (2S)-7,8,bis-3’,4’-(2,2-dimethyl-chromano)-5-hydro
xyflavanone 217, 294, 340 220, 292, 340408 409 221
8 3,7-dihydroxy-4’,8-dimethoxyflavone 250, 304 249, 306 314 315 300
9 14-Hydroxyartonin E 270, 314, 387 269, 315, 387452 453 435

A. Q. LAGHARI ET AL. 876
O
O OH
OH
OH
OH
OH
O
O
O
OMe
O
O OH
OH
OSO
3
H
O
O
OH
OH
OH
Epicatechin (1) (-)-(2S)-6-Methoxy-[2”,3”:7,8]-furanoflavanone (3) Kaempferol 3-O-sulphate-7-O-c-arabinopyranoside (4)
OMe
OH
O
OH
O
O
O OH
O
Vidalenolone (6) (2S)-7,8,bis-3’,4’-(2,2-dimethyl-chromano)-5-hydroxyflavanone (7)
O
O
OH
OH
OMe
OMe
O
OOH
OH
OH
OH
OH
O
3,7-dihydroxy-4’,8-dimethoxyflavone (8) 14-Hydroxyartonin E (9)
Figure 2. Chemical structures of flavonoids and phenolic compounds (1-9) identified from the flowers and leaves.
Table 2. Relative amount of each flavonoid in samples with different extraction techniques.
Extraction techniques
Microwave Soxhlet Marination Sonication Reflux
Compound
Flowers Leaves Flowers Leaves Flowers Leaves Flowers Leaves Flowers Leaves
1 20.3 24.2 18.0 16.2 21.5 16.9 18.7 17.1 21.5 25.6
2 36.9 30.8 18.1 16.3 16.0 17.0 15.4 19.2 13.5 16.7
3 26.9 25.3 15.8 15.7 19.5 21.1 20.3 19.2 17.3 18.6
4 28.5 20.4 17.6 17.8 16.9 21.8 16.2 16.8 20.7 23.2
5 21.5 20.3 19.3 16.9 18.3 20.6 20.0 16.6 20.8 25.7
6 49.2 46.9 50.8 53.1 - - - - - -
7 28.2 25.2 18.0 22.8 18.7 18.6 17.3 16.7 17.8 16.5
8 52.7 - 47.3 - - - - - - -
9 35.7 39.1 18.1 26.6 12.6 15.7 15.6 9.4 17.9 9.1
All values are in relative %.
Table 3. Total flavonoids content in the leaves and flowers and comparison in the extraction methods.
S.NO Extraction methods
Total flavonoids contents in flowers
(mg/g)
Total flavonoids contents in leaves
(mg/g)
1 Microwave extraction 28.15 26.30
2 Soxhlet extraction 27.35 22.52
3 Sonication 20.64 16.41
4 Marinated extraction 22.38 20.83
5 Reflux extraction 18.07 19.91

A. Q. LAGHARI ET AL.877
method and found to be the more efficient for extracting
more number of flavonoids in higher quantity and also
or leaves extracts show highest
DPPH scavenging ability than the reference BHA stan-
avonoids and antioxidative prop
of C. angustifolia were made in present study. Different
present study made by the
Table 4. DPPH % inhibition activity of the extracts of
flowers (a), leaves (b) and reference.
extracts obtained by this method show higher antioxidant
activity. On the other hand the rest of the methods were
very difficult more time consuming and their flavonoids
extraction efficiency was also low as compared to mi-
crowave extraction method. These comparative results
are tabulated in Tables 2-4.
3.4. Assay of DPPH
C. angustifolia flowers
dard (Table 4). Among all extraction techniques, it was
found that microwave extracts show highest antioxidant
activity than rest of the extracts obtained by other tech-
niques. Flowers show high antioxidant activity as com-
pared to leaves. Only 11% of the extract was enough to
exhibit 50% DPPH with reference to BHA in the same
time. Thus, it can be assumed that the contribution to
antioxidant ability of C. angustifolia extracts is mainly
due to the presence of all identified flavonoids, which were
extracted in highest quantity by microwave extraction.
4. Conclusions
Determination of flerty
extraction methods were used to extract the flavonoids
and to find out the best one by comparing their efficiency
and robustness. Among all these methods; microwave
extraction was found to be the best method for extraction
of flavonoids. All extracts show noticeable antioxidant
activity. The importance of C. angustifolia with these
properties, i.e. high antioxidant activity, total flavonoids
content and presence of identified compounds (1-9) make
it an eager and better alternative to the synthetic antioxi-
dant agents and flavonoid sources. The compounds 2 and
5 have not been identified because their data do not
match with reported literature; may be these are new and
in future studies their isolation, identification and bio-
logical activities will be carried out. All the identified
flavonoids are first time reported from this plant, this
data may be useful for the isolation of the targeted iden-
tified flavonoids. The compound 6 is not a flavonoid, but
it is identified as Vidalenolone; a phenolic metabolite.
Hence, the present study may be helpful to pharmaceuti-
cal industry as well as to folk medication practitioners,
who use this plant as a source of curing diseases.
5. Acknowledgements
Financial support during the
S.NO Compound/Sample 1C50 (a) 1C50 (b)
* *
1 BHA 347.2 µM 347.2 µM
2 Microwave 3.1 mg/L 3.6 mg/L
3 Soxhlet
M
3.4 mg/L 4.2 mg/L
4 Sonication 5.9 mg/L 7.4 mg/L
5 arinated3.5 mg/L 5.6 mg/L
6 Reflux 6.5 mg/L 6.2 mg/L
*Conctration (in mh 50% inhiPH by
reference; *Concentration (mg/L) at which 50% inhibition of DPPH is ob-
servey samples in thme period byce.
d Pakistan
ouncil of Scientific and Industrial Research, Kara-
[1] S. Hamamatsu, K. Yabe and Y. Nawa, “Compositions of
d Other Flavonoids in Cultured Rabbiteye
ccinium ashei Reade cv. Tiiblue),” Food
enoles) at whicbition of DPis observed
d be same ti the referen
National Centre of Excellence in Analytical Chemistry,
University of Sindh, Jamshoro/Pakistan an
C
chi/Pakistan is gratefully acknowledged.
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