Industrial Crops and Products 52 (2014) 815–826

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Industrial Crops and Products

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Phytochemical analysis and biological properties of

Cyperus rotundus L.
Kandikattu Hemanth Kumar, Sakina Razack, Ilaiyaraja Nallamuthu, Farhath Khanum ∗
Biochemistry and Nanosciences Discipline, Defence Food Research Laboratory, Mysore, India

a r t i c l e i n f o a b s t r a c t

Article history: Cyperus rotundus is a traditional medicinal herb used in the treatment of various ailments. In the
Received 25 August 2013 present study, we have evaluated in vitro anti-oxidant and free radical scavenging activities of 70%
Received in revised form ethanolic, methanolic and water extracts of C. rotundus root. Among the extracts, the 70% ethano-
19 November 2013
lic extract was found to be most potent with the IC50 values of 64.64 ± 5.3 ␮g/mL, 85.89 ± 6.3 ␮g/mL
Accepted 22 November 2013
and 8.42 ± 0.45 mg/mL against DPPH, metal chelating and nitric oxide scavenging activities and the
observed activity could be correlated with metabolites such as polyphenols, flavonoids and sesquiter-
Keywords:
penes identified by LC–ESI-MS/MS. Further C. rotundus was evaluated for its protective properties against
Cyperus rotundus
LC–ESI-MS/MS
macromolecules such as DNA, protein and lipid damage. The extract showed 48% protection against H2 O2
Oxidative stress induced DNA damage and inhibited the AAPH and SIN-1 induced oxidation and nitration of BSA. More-
Acetylcholine esterase activity over, C. rotundus pretreatment restored the antioxidant status in white blood cells treated with H2 O2 . C.
DNA damage rotundus showed acetylcholine esterase inhibitory activity and the extract was found to be non-toxic up
Anxiolyte to 100 ␮g/mL in SH-SY5Y human neuroblastoma cell line. The ethanolic extract (200 mg/kg) also proved
to be a potent anxiolyte as assessed by behavioural tests. Overall, the results suggest that this plant extract
may be useful in combating oxidative stress related diseases through its antioxidant activity.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction as carotenoids, tocopherols and flavonoids prevent and protect the

cells from oxidative stress related diseases (Vitaglione et al., 2004;
Reactive oxygen species (ROS)/free radicals such as superoxide Tai et al., 2005). Therefore, it is worthwhile to explore the natural
anion radicals (O2 − ), hydroxyl radicals (OH·), and non-free radical antioxidants and their mechanism of action at cellular levels.
species such as hydrogen peroxide (H2 O2 ) and singlet oxygen (O2 ·) Cyperus rotundus is a traditional medicinal herb belongs to
are produced as part of normal body’s functions like respiration Cyperaceae family and is widespread in India. The major chemical
and metabolic activities. These free radical generation leads to per- constituents of this herb include essential oils, flavonoids, ter-
oxidation of lipids, which in turn stimulates glycation, oxidation, penoids, sesquiterpenes, sitosterol, cyperene, cyperol, nootkatone
nitration of proteins and inactivation of enzymes, DNA damage and valencene (Sonwa and König, 2001; Tsoyi et al., 2011). In an
and other alterations in the cellular organelles (Finkel, 2003). All earlier study Thebtaranonth et al. (1995) had demonstrated the
the aerobic organisms have multiple antioxidant defense mecha- antimalarial activity of sesquiterpene 4,7-dimethyl-1-tetralone.
nisms and most importantly the enzymatic oxidant system that Jeong et al. (2000) had isolated three novel sesquiterpene alka-
include anti-oxidant enzymes such as superoxide dismutase (SOD), loids such as Rotundines A, B and C, from C. rotundus. Chen et al.
catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (2011) described the analgesic activity of C. rotundus and also ana-
(GR) and glucose 6-phosphate dehydrogenase (G-6-PDH). The free lyzed its active constituents by GC–MS which shows the presence
radical induced oxidative damage has been implicated in several of ␣-cyperone. Jung et al. (2013) reported that ␣-cyperone, inhibits
neuronal disorders like Alzheimers, Parkinsons and Amyotrophic LPS-induced inflammation in RAW 264.7 cells by regulation of COX-
lateral sclerosis (Andersen, 2004). Supplementation of antioxidants 2 expression, PGE2 production and NF␬B signalling. The rhizome
can therefore protect the human body from the deleterious effect of of this plant has been reported to have analgesic, nootropic, seda-
these radicals. Herbal remedies have been used for centuries to cure tive, antimalarial, anti-inflammatory and antispasmodic properties
various ailments as a source for natural antioxidants. Several lines and also used to relieve diarrhoea and bowel disorders (Zhu et al.,
of evidences also have demonstrated that natural antioxidants such 1998; Uddin et al., 2006; Kilani et al., 2005; Tsoyi et al., 2011). The
starch extracted from tuberous part of this plant found to have var-
ious applications in food and confectionary industries (Umerie and
∗ Corresponding author. Tel.: +91 821 2474676; fax: +91 821 2473468. Ezeuzo, 2000). Of late, Lee et al. (2010) and Sunil et al. (2011) have
E-mail address: [email protected] (F. Khanum). demonstrated the neuroprotective role of C. rotundus in in vitro

0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.11.040
816 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826

Parkinsonism and in vivo cerebral ischaemia models. Recently, we The flavonoid content was determined according to the method
also demonstrated the anti-apoptotic activity of C. rotundus using described by Delcour and Varebeke (1985). To 1 mL of appropriately
SH-SY5Y human neurons (Kumar and Khanum, 2013; Hemanth diluted extracts, 5 mL of chromogen reagent (0.1% cinnamaldehyde
Kumar et al., 2013). solution in a cooled mixture of 75 mL methanol and 25 mL concen-
The objectives of the present study were to evaluate the antiox- trated HCl) was added. After an incubation period of 10 min, the
idant, free radical scavenging, macromolecular damage protective, absorbance was recorded at 640 nm. The total flavonoid content
acetylcholine esterase inhibitory, anxiolytic and cytotoxic effects was expressed in ␮g catechin equivalents (CE)/mg extract.
of C. rotundus.
2.5. In vitro antioxidant and free radical scavenging activities
2. Materials and methods
2.5.1. DPPH radical scavenging activity
2.1. Chemicals and reagents The radical-scavenging activity or hydrogen-donating ability
of the extracts was studied using the stable DPPH* radical, as
Acetylcholineesterase, eserine, DTNB (5,5 -dithiobis(2-nitr- described by Blois (1958) with slight modifications. Briefly, to
obenzoic acid)), gallic acid, ascorbic acid, EDTA (ethylenedia- 3 mL of appropriately diluted extract in methanol, 0.5 mL of DPPH*
minetetraacetic acid), AAPH (2,2 -azobis(2-methylpropio- (500 ␮M) added and the reaction mixture was allowed to stand in
namidine) dihydrochloride), SIN-1 (3-morpholinosydnonimine), dark for 45 min at room temperature and absorbance was recorded
Dulbecco’s Modified Eagle’s Medium: Ham’s nutrients mix F-12 at 515 nm against the blank. A sample control reading was recorded
(1:1, DMEM/F-12), foetal bovine serum (FBS), glutamine, penicillin, without plant extracts under identical conditions. The percentage
streptomycin solution and MTT (3-(4,5-dimethyl-2-thiazolyl)- of free radical scavenging capacity of the extracts was calculated
2,5-diphenyl-2H-tetrazolium bromide) were obtained from and expressed as IC50 values. BHA was used as a standard antioxi-
Sigma–Aldrich (MO, USA). DPPH (2,2-diphenyl-1-picrylhydrazyl), dant.
ammonium molybdate, ferrozine, ABTS (2,2 -azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt), FC
2.5.2. Metal chelating activity
(Folin-Ciocalteu) reagent, BHA (butylhydroxyanisole) were of
The chelating effect of extracts on ferrous ions was estimated
Hi-media and Merck brands (Bangalore, India). All other chemicals
as per the method described by Dinis et al. (1994). Briefly, 2 mL of
were of analytical grade and obtained from Rankem (Bangalore,
appropriately diluted extracts were mixed with 0.05 mL of 2 mM
India).
FeCl2 and the reaction was initiated by the addition of 0.2 mL of
5 mM ferrozine and incubated for 10 min at room temperature. The
2.2. Preparation of C. rotundus extracts
absorbance of the mixture was measured spectrophotometrically
at 562 nm. The percentage inhibition of ferrozine–Fe2+ complex
The roots of C. rotundus, obtained from the local market of
formation was calculated and the results were expressed in IC50
Mysore, India, were shade dried and finely powdered. Three dif-
values. EDTA was used as a standard antioxidant.
ferent extracts were prepared using methanol, 70% ethanol and
water with the solvent ratio of 1:10 (w/v). The extracts were con-
centrated by flash evaporator (Rotavac, Schwabach, Germany) and 2.5.3. ABTS radical scavenging activity
freeze dried using lyophilizer (Lyolab, Hyderabad, India) as per the The ABTS (2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic
requirements. acid) cation radical scavenging activity was performed with slight
modifications as described by Re et al. (1999). The assay is based
2.3. LC–ESI-MS/MS analysis of hydroalcoholic fraction of C. on the principle of quenching the ABTS•+ by antioxidant com-
rotundus pounds. The ABTS•+ radicals were generated by mixing 7 mM
ABTS and 2.45 mM potassium persulphate. Prior to use, the solu-
LC–ESI-MS/MS analysis of 70% ethanolic fraction of C. rotun- tion was diluted with ethanol to get a blue-green chromophore
dus was performed on 6520 Accurate Q-TOF (Agilent Santa with an absorbance of 0.700 ± 0.025 at 734 nm. For the assay
Clara, CA) mass spectrometer coupled to HPLC equipped with 10 ␮L of test sample was mixed with 1.0 mL of ABTS solution in
UV–vis detector. The column was Zorbax SB C18 Rapid resolution, a microcuvette. The decrease in absorbance was quantified spec-
4.6 mm × 150 mm, 3.5 ␮ particle size m and the conditions were: trophotometrically after 6 min of incubation at room temperature.
(A) formic acid (0.1%, v/v) and 10 mM ammonium phosphate (B) The extent of decolourization was calculated as percentage reduc-
acetonitrile + 0.1% formic acid; gradient (in solvent B): (i) 35%, from tion of absorbance by the extract and gallic acid standard. The
0 to 0.5 min, (ii) 55%, from 10 min (iii) 95%, from 25 to 33 min (iv) ABTS•+ concentration (mM) in the reaction medium was calculated
35%, at 35–40 min of total run time; flow rate: 0.2 mL/min; injec- from calibration curve.
tion volume 3 ␮L; ESI parameters: both negative and positive ion
mode; mass range 100–1200 m/z; spray voltage 4 kV; gas temper- 2.5.4. Nitric oxide scavenging activity
ature 325 ◦ C; gas flow 10 L/min; Nebulizer 40 psi. The assay is based on the principle that sodium nitroprusside
in aqueous solution at neutral pH spontaneously generates nitric
2.4. Total phenol and flavonoid contents oxide, which in turn reacts with oxygen to produce nitrite ions that
can be estimated using Greiss reagent. Scavengers of nitric oxide
The total phenolic contents of the extracts were determined compete with oxygen molecules that result in reduced production
using FC reagent, where gallic acid was used as a standard antiox- of nitrite ions. In the experiment, sodium nitroprusside (10 mM)
idant (Kujala et al., 2000). The results were expressed in ␮g gallic in phosphate buffered saline was mixed with different concen-
acid equivalent (GAE)/mg extract. To 3 mL of appropriately diluted trations of samples/standard followed by incubation at room
extract, 0.5 mL of FCR was added, followed by incubation at room temperature for 150 min. The same reaction mixture without the
temperature (10 min) and addition of 7% Na2 CO3 (2 mL) solution. sample/standard served as the control. After the incubation period,
The mixture was boiled for 1 min and absorbance of the colour was 0.5 mL of Griess reagent [1% sulfanilamide, 2% H3PO4 and 0.1% N-
recorded at 750 nm using spectrophotometer (Shimadzu, Kyoto, (1-naphthyl) ethylenediamine HCl] was added and the absorbance
Japan). of the formed chromophore by the diazotization of nitrite with
 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826 817

sulphanilamide and subsequent coupling with naphthylethylene- 2.7. Protective effect on oxidation of biomolecules ex vivo
diamine dihydrochloride is measured spectrophotometrically at
546 nm (Sreejayan and Rao, 1997). The percent scavenging activ- 2.7.1. Isolation of lymphocytes and treatments
ity of the nitric oxide generated was measured by comparing the White blood cells (WBC) were isolated from rat blood using
absorbance values of control and test preparations and the results HiSep LSM LS001 (HIMEDIA) according to the manufactures
were expressed in terms of IC50 values. l-ascorbic acid was used as instructions. Briefly, anticoagulated blood was diluted with PBS
a reference standard. (0.01 M, pH 7.2) and overlayed on the HiSep LSM and centrifuged at
400 × g at room temperature for 15 min. The lymphocyte layer was
aspirated and washed twice with PBS at 200 × g for 3 min at 4 ◦ C and
2.5.5. Ferric reducing antioxidant power (FRAP) assay finally diluted to 5 × 106 cells/mL. Cells incubated with PBS alone
This assay was used to estimate the reducing capacity of C. served as control group. In another two groups, cells were treated
rotundus extracts, according to the method described by Benzie with 100 ␮M H2 O2 for 5 min on ice with or without pretreatment
and Strain (1996). The principle of the assay is based on the reduc- of 50 ␮g/mL of C. rotundus for about 30 min. After the treatments,
tion of (Fe3+ ) ferricyanide in stoichiometric excess relative to the cells were centrifuged and washed twice with PBS at 200 × g for
antioxidant potential of the extracts. The FRAP reagent contained 3 min at 4 ◦ C. These cells were used for estimation of DNA damage
2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM and antioxidant enzyme levels.
FeCl3 • 6H2 O and 25 mL of 300 mM acetate buffer (pH 3.6). To 900 ␮L
FRAP reagent 70 ␮L water and 30 ␮L of the extracts were added. 2.7.2. Single cell gel electrophoresis assay (SCGE)
The reaction mixture was incubated at 37 ◦ C for 30 min. Finally, The integrity of DNA against H2 O2 induced oxidative stress was
the absorbance was measured at 593 nm and the results were evaluated by alkaline comet assay according to the method of Singh
expressed in IC50 values. et al. (1988). The lymphocytes (4 × 105 cells/mL) were mixed with
100 ␮L of 0.7% (w/v) low melting agarose (LMA) and loaded to the
frosted slides pre-coated with 1.0% (w/v) normal melting agarose.
2.6. Protective effect on oxidation of biomolecules in vitro
Once the agarose set, the slides were covered with another 100 ␮L
of 0.7% (w/v) LMA and to lyse the cells and nuclear membranes
2.6.1. Plasmid DNA strand break assay
the slides were immersed in freshly prepared cold lysis buffer
The protective efficacy of ethanolic extract against the H2 O2
(2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl buffer, 0.1% (v/v) SDS,
induced DNA damage was studied using pRSET-A plasmid DNA
and 1% (v/v) Triton X-100 and 10% dimethyl sulfoxide; pH 10.0)
(Russo et al., 2000). Briefly, to 350 ng of plasmid DNA in 10 ␮L of TE
for 90 min. Later, the slides were transferred to an electrophore-
buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0), H2 O2 was added
sis tank, and incubated in alkali buffer (3 M NaOH, 10 mM EDTA;
to a final concentration of 10 mM with or without C. rotundus (10, 25
pH 13.0) for unwinding the DNA followed by electrophoresis with
and 50 ␮g/mL) and incubated for a period of 5 min at room temper-
an electric current of 25 V/300 mA for 20 min. Further, the slides
ature. Plasmid DNA without any extract was used as a control. After
were washed twice with neutralizing buffer (0.4 M Tris–HCl, pH
treatments, the plasmid DNA was electrophoresed on 1% agarose
7.5) for 10 min and treated with ethanol for another 5 min followed
gel. Finally the gel was stained with ethidium bromide and pho-
by staining with 40 ␮L of ethidium bromide (20 ␮g/mL). Finally
tographed using gel documentation system (Syngene, Cambridge,
the DNA damage was evaluated with fluorescence microscope
UK).
(Olympus microscope, Germany) by measuring the percentage of
fluorescence in tail using RS image software and the results were
2.6.2. Protein oxidation expressed as per cent inhibition of tail length.
The oxidation of bovine serum albumin (BSA) was carried out by
an azo compound AAPH, which upon decomposition with oxygen 2.7.3. Estimation of antioxidant status
generates peroxyl radicals (Mayo et al., 2003). Briefly, BSA (5 ␮g) The activity of antioxidant enzymes such as superoxide dismu-
was dissolved in phosphate buffer (pH 7.3) and incubated for 2 h tase (SOD), glutathione peroxidase (GPx), glutathione reductase
with or without AAPH (40 mM final concentration) and in the pres- (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) was
ence or absence of C. rotundus extract. After due incubation, the measured in WBC according to the kit protocols. (Randox, Cat. no.
protein samples were subjected to SDS-PAGE electrophoresis. The SD. 125, RS 504, GR 2368, PD 410, Canada). Catalase (CAT) activity
gels were stained with 0.15% Coomassie Brilliant Blue R-250 and was estimated by measuring the decay of 6 mM H2 O2 solution at
the amount of protein damage was quantified by measuring the 240 nm (Cohen et al., 1970).
density of each band.
2.7.4. Anti-lipid peroxidation activity
The lipid peroxidation products were quantitatively estimated
2.6.3. Nitration of bovine serum albumin in terms of thiobarbituric acid reactive substances (TBARS) formed
BSA (30 ␮g) was incubated in the absence (control) and pres- in the liver homogenate treated with AAPH radicals according to the
ence of SIN-1 (final concentration 37.5 mM) with or without the method of Wright et al. (1981). Liver tissues were collected from
addition of C. rotundus. After 3 h incubation at room tempera- young (3–4 months old, 120–130 g) male Wistar albino rats and
ture, the samples were run on 10% SDS-PAGE and transferred to washed with 0.95% NaCl solution. Tissue homogenates were pre-
nitrocellulose membrane (Millipore, MA, USA). The membrane was pared in ice-cold 3 mM Tris buffer containing 250 mM sucrose and
blocked overnight at 4 ◦ C with 5% (v/v) non-fat dry milk in Tris- 0.1 mM EDTA (pH 7.4). The reaction mixture contained 0.5 mL liver
buffered saline with Tween-20 (TBS-T) (10 mM Tris–HCl, 150 mM homogenate (10%, w/v) and 0–100 ␮L extract and made up to 1 mL
NaCl, and 0.1% Tween-20, pH 7.5). Further the membrane was incu- with phosphate buffer (0.1 M, pH 7.4). Peroxidation was initiated
bated for 3 h with 3-nitrotyrosine antibody (3-NT, N5538, Sigma, by adding 1 mL of 200 ␮M AAPH followed by incubation at 37 ◦ C
St. Louis, MO, USA) and washed with TBS-T buffer followed by for 2 h. The reaction was terminated by the addition of 1.0 mL TCA
incubation with goat anti-mouse peroxidase-labelled secondary (10%, w/v). Then, 1.0 mL of TBA (0.67%, w/v) was added and tubes
antibody (Dako, Denmark) and washing with TBS-T buffer. Finally were placed in a boiling water bath for 20 min. Samples were cen-
the immunoreactivity was detected by chemiluminescenet perox- trifuged at 2500 × g for 10 min and the amount of malondialdehyde
idase substrate (Sigma, USA). formed in each sample was assessed by measuring the absorbance
818 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826

of supernatant at 535 nm against a reagent blank. BHA was used as 3.2.4. Thirsty rat conflict paradigm
a standard antioxidant. This behavioural test was performed according to the method
of Vogel et al. (1971), with slight modifications. Briefly, the
mice were water deprived for 48 h before testing. After 24 h of
3. Neuroprotective effects
water deprivation the mice were placed in test apparatus and
allowed to locate the metallic drinking spout of the water bot-
3.1. Acetylcholine esterase activity
tle. After 48 h of water deprivation the treated animals were
then individually placed in the cage for 3 min, during which
Acetylcholine esterase (AChE) inhibitory activity was measured
the mice received an electric shock (0.5 mA, 1 s) on each con-
according to the spectrophotometric method developed by Ellman
tact with the metallic spout. The total number of drink contacts
et al. (1961). Briefly, 1.25 mL of 100 mM sodium phosphate buffer
and the shocks accepted during this test period of 3 min was
(pH 8.0), 50 ␮L of sample solution at different concentrations (125,
recorded.
250, 375, 500, 625 and 750 ␮g/mL), 5 ␮L of diluted AChE and 20 ␮L
of acetylthiocholine iodide (75 mM) were mixed and incubated
3.3. Cytotoxic effect of C. rotundus in cell culture
for 15 min at room temperature. The hydrolysis of substrate was
monitored spectrophotometrically by the estimation of yellow 5-
The human neuroblastoma SH-SY5Y cell line was obtained
thio-2-nitrobenzoate anion, after adding 100 ␮L of 10 mM DTNB
from the National Centre for Cell Sciences, Pune, India. Cells were
at a wavelength of 412 nm. The experiments were carried out in
seeded into 96 and 24-well plates in 1:1 mixture of DMEM/F-
triplicate and the per cent AChE inhibition was calculated in com-
12 supplemented with 10% FBS, 2 mM l-glutamine, antibiotic and
parison with acetylcholine esterase inhibitory activity of eserine
antimycotic solution in a humid atmosphere of 5% CO2 and 95% air
(standard).
at 37 ◦ C. The media was changed on alternative days and once the
confluency was reached, the cells were treated with C. rotundus for
3.2. Analysis of anxiolytic activities of C. rotundus by behavioural 24 h at different concentrations (10, 25, 50, 100, 150, 200, 400, 500
tests and 1000 ␮g/mL).

Male albino (15 weeks) mice of Balb/C strain weighing 25 ± 3.0 g 3.3.1. MTT assay
were selected from the stock colony, Defence Food Research The mitochondrial health of human neurons after treatment
Laboratory, Mysore, India, housed in an acrylic fibre cage in a tem- with C. rotundus was assessed by MTT assay. The principle of the
perature controlled room (temperature 25 ± 2 ◦ C) and maintained assay is based on the cleavage of tetrazolium salts by mitochon-
in 12 h light (6 am–6 pm)/dark cycle (6 pm–6 am). Food and water drial succinate reductase in viable cells to form formazan dye.
were provided ad libitum and the animals were acclimatized for 7 The SH-SY5Y cells were seeded in 96-well plates at a density of
days prior to dosing. Thirty-two mice were randomly divided into 1 × 104 cells/well and grown for 24 h and then subjected with dif-
the following four experimental groups with eight animals in each ferent doses of C. rotundus (10–1000 ␮g/mL). After treatments, MTT
group. (I) Control group (normal saline), (II) C. rotundus 100 mg/kg (0.5 mg/mL) was added to each well and incubated for 2 h at 37 ◦ C
bw (body weight) (III) C. rotundus 200 mg/kg bw ( IV) Diazepam and the formed formazan crystals were dissolved in DMSO. The
1 mg/kg bw which were administered by gavage for a period of 7 absorbance was then measured at 540 nm using a VERSA max Hidex
days. Body weights were recorded during the experimental period. plate chameleonTM V (Finland) and the % inhibition of cell viability
Institute Animal Ethical Committee (IAEC) and Committee for the was expressed.
Purpose of the Control and Supervision of Experiments on Animals
(CPCSEA) approved the protocol for animal behavioural studies.
3.3.2. Lactate dehydrogenase (LDH) release assay
Cytotoxic effect of the C. rotundus extract on the SH-SY5Y cells
3.2.1. Open field test was studied by measuring the amount of LDH released in to the
The apparatus consisted of a brightly lit arena measuring medium by means of an LDH-estimation kit (Agappe-11407002,
60 cm × 20 cm divided into 24 equal squares. The animals were Mysore, India). LDH catalyses the oxidation of lactate to pyruvate
orally fed with C. rotundus and Diazepam 60 min before the start with simultaneous reduction of nicotinamide adenine dinucleo-
of the test. The animals were placed in the centre of the apparatus tide (NAD+ ) which can be measured at a wavelength of 340 nm.
and the total locomotor activity was recorded as the number of line The rate of increase in enzyme activity is due to the formation of
crossings for 5 min (Kulkarni, 2005). reduced nicotinamide adenine dinucleotide (NADH) that is directly
proportional to the LDH activity in the sample. The SH-SY5Y cells
were plated at a density of 5 × 104 cells/well in 24-well plates.
3.2.2. Elevated plus maze test After 24 h, the cells were treated with 10 ␮g to 1 g of C. rotundus
The elevated plus maze consisted of two open arms 35 cm × 5 cm for 24 h and then lysed with, 10 ␮L of 2% Triton X-100. Untreated
crossed with closed arms 35 cm × 5 cm × 15 cm with a central plat- cells were used to measure the total LDH activity. The cells were
form measuring 5 cm × 5 cm. The whole apparatus was elevated to precipitated by centrifugation at 2500 rpm for 5 min at 4 ◦ C. The
a height of 60 cm from the floor level. The animals, with respective supernatant (100 ␮L) was mixed with 900 ␮L of kit reaction mix-
drug treatment (as mentioned above), were placed in the centre of ture and the enzyme activity was measured in terms of intracellular
the maze, facing one of the open arms and observed for the number LDH released into the medium.
of entries and time spent in each arm, thereafter for 5 min (Lister,
1987).
3.4. Statistical analysis

3.2.3. Light–dark box test The results were analyzed by one-way ANOVA. Significance was
The light–dark box consists of a small dark secure compartment set at 0.05 and all comparisons were made against the control group
(15 cm × 20 cm) and a large illuminated aversive compartment for behavioural tests of anxiety experiments. Whereas other tests
30 cm × 20 cm. The number of entries and time spent in light–dark were performed in triplicate and mean ± standard deviation was
box by animals was recorded for 5 min (Bourin and Hascoët, 2003). expressed.
 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826 819

Fig. 1. LC–ESI-MS/MS of hydroalcoholic fraction of Cyperus rotundus.

4. Results 4.3. In vitro antioxidant and free radical scavenging activities

4.1. Phytochemical analysis 4.3.1. DPPH radical scavenging activity

The results of the present study shows that a dose dependent
Phytochemical analysis was carried out by LC-Q-TOF/MS to increase in quenching of free radical with an increase in extract con-
obtain the metabolite profiles of hydroalcoholic fraction of C. centration. 70% ethanolic extract was found to be most potent with
rotundus. The identity of compounds was confirmed by mass frag- the lowest IC50 value followed by methanolic and water extracts
mentation analysis and the MS–MS spectra of the compounds are as shown in Table 2. The data obtained clearly indicate that the
shown in Fig. 1. Positive ion mode detected 15 metabolites where increased DPPH scavenging ability of the alcoholic extract may be
as negative ion mode detected 6 metabolites, a total of 22 metabo- attributed to its potent hydrogen donating ability.
lites were identified (see Supplementary Material for TIC and HPLC
chromatograms).

Table 1
Estimation of total phenols and flavonoids.
4.2. Total phenolic and flavonoid content
Extract Total phenolic content Total flavonoids
The polyphenol contents were found to be in the (␮g GAE/mg extract) (␮g CE/mg extract)
decreasing order of water followed by methanolic and 70% 70% Ethanol extract 254.50 ± 5.26 164.34 ± 3.75
ethanolic extracts 70.75 ± 4.48 < 192.77 ± 5.48 < 254.5 ± 5.26 Methanol extract 192.77 ± 5.48 138.01 ± 5.24
and the same trend was observed with flavonoid content Water extract 70.75 ± 4.48 51.23 ± 2.62
51.23 ± 2.62 < 138.01 ± 5.24 < 164.34 ± 3.75 (Table 1). Values are represented as means ± SD of triplicate determination.
820 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826

Table 2
(A) Estimation of antioxidant and free radical scavenging activity.

Assay IC50 value (␮g/mL) 70% Ethanol extract Methanol extract Water extract

DPPH 64.64 ± 5.3 86.17 ± 3.6 300.62 ± 5.2

Metal chelation 85.89 ± 6.3 98.51 ± 7.2 650 ± 16.8
ABTS 15.49 ± 1.9 15.42 ± 1.9 152.6 ± 6.5
FRAP 52.78 ± 4.9 87.79 ± 7.2 112.55 ± 11.2
NO scavenging 8.42 ± 0.45 9.39 ± 0.5 15.98 ± 0.6
Anti-lipid peroxidation 503.48 ± 18.2 545.39 ± 16.4 894.59 ± 22.2

Values are represented as means ± SD of triplicate determination.

4.3.2. Metal chelation 4.3.4. FRAP assay

The method for this activity is based on chelating of Fe+2 by Ferric reducing antioxidant power is a powerful in vitro assay,
the reagent ferrozine, gives quantitative decrease of ferrous and which is used in determination of antioxidant activity based on
ferrozine complex formation. The chelating activity was found to reduction of ferric iron (III) into ferrous iron (II). The antioxidant
be in the decreasing order of 70% ethanolic, methanolic and water activity of 70% ethanolic, methanolic and water extracts were found
extracts with IC50 of 85.89 ± 6.3, 98.51 ± 7.2, 650 ± 16.8 ␮g/mL to be 74.85 ± 6.9, 112.23 ± 10.5, 619.12 ± 21.3 ␮g/mL, respectively,
respectively (Table 2). which indicates the potential antioxidant status of 70% ethanolic
extract followed by methanolic and water fractions (Table 2).

4.3.3. ABTS radical scavenging activity

ABTS assay is based on the principle of the amount of extract
required to scavenge 50% of ABTS•+ radical via the mechanism 4.3.5. Nitric oxide scavenging activity
of electron-/hydrogen-donation. Table 2 depicts the ABTS radical Incubation of sodium nitroprusside in PBS at room tempera-
scavenging activity of C. rotundus extracts. Among the extracts the ture resulted in linear time-dependent nitrite production which
70% ethanolic extract was found to be the most effective radical is remarkably reduced by all the extracts of C. rotundus. The
scavenger followed by methanolic and water fractions. ethanolic extract showed better NO scavenging activity with IC50

Fig. 2. The protective effect of C. rotundus on H2 O2 induced plasmid DNA damage analysis by agarose gel electrophoresis. (B.i) The protective effect of C. rotundus on AAPH
induced protein oxidation of BSA analyzed by polyacrylamide gel electrophoresis. (B.ii) The densitometric analysis of protein oxidation by NIH Image J software. (C.i) The
protective effect of C. rotundus on SIN-1 induced expression of protein nitration biomarker 3-NT analyzed by westernblotting. (B.ii) The densitometric analysis of individual
bands were carried out by NIH Image J software.
 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826 821

of 8.42 ± 0.45 mg/mL followed by methanolic and water extracts analysis is presented in Fig. 2B.ii. The azo compound AAPH induced
(Table 2). the oxidative degradation of BSA. After 2 h incubation with 40 mM
The results of the present study showed that 70% ethanolic AAPH (positive control), the density of BSA band decreased to
extract is the most potent scavenger of free radicals compared to 46.5% compared to negative control while the pretreatment of BSA
other fractions and therefore it was selected for further studies. with 25 and 50 ␮g C. rotundus completely prevented this oxidative
degradation of BSA (Fig. 2B.i).
4.4. Protective effect on oxidation of biomolecules in vitro
4.4.3. Inhibitory activity of C. rotundus on protein nitration
4.4.1. Prokaryotic DNA damage preventing efficacy of C. rotundus In the present study, SIN-1 was used to induce protein nitration
by agarose gel electrophoresis and the extent of nitration was detected by immunoreactivity with
The 70% ethanolic extract was evaluated for its protective effect 3-NT antibody. Pretreatment of BSA with 25 and 50 ␮g/mL of C.
on H2 O2 induced damage of pRSET-A plasmid DNA. The agarose gel rotundus dose dependently inhibited the protein nitration, while no
pattern of DNA with in the presence of 10, 25 and 50 ␮g of C. rotun- immunoreactivity was observed in BSA alone group which served
dus is shown in Fig. 2A. Negative control (lane-1) showed two bands as a control (Fig. 2C.i and ii).
on agarose gel electrophoresis wherein the faster moving band cor-
responded to the native supercoiled circular DNA (ScDNA) and the 4.5. Protective effect on oxidation of biomolecules ex vivo
slow moving faint band corresponded to the open circular form
(OcDNA). Addition of H2 O2 (lane 2) resulted in conversion of super- 4.5.1. Eukaryotic DNA damage preventing efficacy of C. rotundus
coiled DNA to linear form indicating the hydroxyl radical mediated by SCGE
DNA damage. Whereas treatment with C. rotundus provided sig- The protective effect of C. rotundus against H2 O2 induced
nificant protection to the damage of ScDNA in a dose dependent genomic DNA damage in WBC extracted from rat blood was ana-
manner (lanes 3–5). lyzed by SCGE. The principle of the assay is undamaged DNA being
too large retains in a highly organized association with protein
4.4.2. Protective efficacy of C. rotundus on protein oxidation matrix in the nucleus, whereas this organization is disrupted in
Electrophoretic patterns of BSA exposed to AAPH in the pres- damaged DNA. When electric field is applied, the damaged DNA
ence or absence of C. rotundus and the corresponding densitometric fragments move faster in gel and are visualized in the form of a

Fig. 3. (A) Effect of C. rotundus on DNA damage induced by H2 O2 in WBC. Control cells without any treatment (a), DNA damage induced by treatment with 100 ␮M H2 O2 (b),
SH-SY5Y cells were pre-treated with C. rotundus for 2 h at 50 ␮g/reaction and induced with 100 ␮M H2 O2 (c). (B) Tail length (␮m): bars, inhibitory effect of C. rotundus on
cell damage: diamond.
822 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826

comet, whereas undamaged DNA strands, being too large, do not 4.7.2. Elevated plus maze test
leave the cavity. The tail length of comet is used as an index to Mice administered with C. rotundus at 100 and 200 mg/kg bw
measure the extent of DNA damage in the cell. spent more time on the open arms and also significantly more
We observed increased fluorescence intensity in tail of the cells number of entries were observed into open arms of the plus maze
exposed to 100 ␮M H2 O2 which induced the DNA damage, whereas (p < 0.05) in comparison with control (Fig. 5B).
the tail fluorescence intensity was decreased in the cells pre-treated
with C. rotundus (Fig. 3A and B). The extract at a concentration of 4.7.3. Light–dark box test
50 ␮g/reaction showed 51.3% inhibitory effect against cell damage, C. rotundus at 100 and 200 mg/kg bw significantly increased
as measured in terms of tail length. The results clearly demonstrate number of entries as well as the time spent in the light box (p < 0.05)
that H2 O2 -induced DNA damage was successfully repaired by the of the test apparatus. This suggests that the plant extract at this dose
active principles present in C. rotundus. is working effectively as an anxiolyte. Similarly diazepam also sig-
nificantly increased (p < 0.05) the time spent in the light box of the
4.5.2. Estimation of antioxidant enzyme levels apparatus (Fig. 5C).
In the current study, we investigated the antioxidant activity
of C. rotundus against the H2 O2 challenge of WBC. H2 O2 -treated 4.7.4. Thirsty rat conflict paradigm
cells resulted in decreased activities of SOD, CAT, GPx and GR while In the present study, a significant increase (p < 0.05) in punished
C. rotundus pre-treatment restored the antioxidant activities of all drinking behaviour was observed in C. rotundus treated group in
these enzymes (Table 3). comparison with control (Fig. 5D). Diazepam similarly increased
the number of licks and shocks accepted.
4.5.3. Anti-lipid peroxidation activity
The inhibition of lipid peroxidation induced by AAPH in rat liver 4.8. Cytotoxicity of the plant extract
homogenate was assayed spectrophotometrically by measuring the
TBARS. All the extracts of C. rotundus inhibited the lipid peroxi- The SH-SY5Y cells were treated with different concentrations of
dation in a dose dependent manner and the corresponding IC50 C. rotundus (10, 25, 50, 100, 150, 200, 400, 500 and 1000 ␮g/mL) for
values were shown in Table 2. The results showed that maximum 24 h and the cell viability was determined by MTT and LDH assays.
lipid peroxidation inhibitory activity was exhibited by 70% ethano- The viability of SH-SY5Y cells was found to be same as untreated
lic extract with lowest IC50 value of 503.48 ± 18.2 ␮g/mL followed control cells up to 100 ␮g/mL concentration. However, a signifi-
by methanol and water extracts. cant decrease in cell viability and LDH leakage was observed above
100 ␮g/mL C. rotundus treatment (Fig. 6A and B).
4.6. Neuroprotective effects of C. rotundus
5. Discussion
4.6.1. Acetylcholineesterase activity
In the present study, we observed that 70% ethanolic extract Free radical-mediated oxidation of biological molecules such as
of C. rotundus inhibited the activity of electric eel AChE in a dose lipids, proteins, and DNA has been implicated in a variety of dis-
dependent manner and showed maximum inhibition of AChE at a orders and diseases (Wiseman and Halliwell, 1996). Consequently,
concentration of 500 ␮g (Fig. 4). the role of free radical-scavenging activity of antioxidants has been
widely studied through various experiments. These antioxidants
4.7. Anxiolytic effects of C. rotundus are known to be abundant in fruits, vegetables, and beverages
(Wootton-Beard, 2011). The potential role of natural antioxidant
4.7.1. Open field test nutrients includes prevention of cancer, cardiovascular diseases,
Supplementation of mice with C. rotundus at 100 and 200 mg/kg neurodegenerative diseases, and ageing (Liu, 2004; Yang et al.,
body weight (bw) significantly alleviated anxiety symptoms 2009). Furthermore, studies on the effectiveness of natural antiox-
(p < 0.05) by increasing the total locomotor activity as assessed by idants are required to understand and utilize the biological action
the number of lines crossed against control. The results were similar of natural antioxidants at cellular level.
for diazepam treated group (Fig. 5A). The phenolics and flavonoids are the major groups of secondary
metabolites which act as primary antioxidants and free radical ter-
minators. Flavonoids, being one of the most diverse and widespread
80 group of natural compounds, are likely to be the most abun-
Per cent AChE inhibition

dant and important natural phenolics. Therefore, in our present

64 investigation, the extracts were analyzed for total phenolic and
flavonoid contents. The phenolic compounds such as 3-hydroxy-4-
methoxy-benzoic acid, galloyl quinic acid, ferulic acid and flavonoid
48 compounds such as quercetin, luteolin, afzelechin, catechin are
the principle antioxidants present in C. rotundus as reported by
32 Kilani-Jaziri et al. (2009). It has been reported that total oligomeric
flavonoid extract of C. rotundus possess a broad spectrum of
pharmacological properties such as antioxidant, antimutagenic,
16
antigenotoxic, antimicrobial, anticancer and neuroprotective prop-
erties (Kilani et al., 2005; Kilani-Jaziri et al., 2009, 2011; Sunil et al.,
0 2011). In the present study, we performed an array of in vitro
0 0.125 0.25 0.375 0.5 0.625 0.75 antioxidant and free radical scavenging activities to evaluate the
scavenging ability of C. rotundus fractions against various free rad-
Concentration ( g/ml)
icals.
Fig. 4. Acetylcholineesterase inhibitory activity of C. rotundus. The results are rep- The bleaching of DPPH* is used as a representative of free rad-
resented as mean± of three independent experiments. ical or antioxidant scavenging activity by plant extracts as well as
 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826 823

Table 3
Estimation of antioxidant enzymes in WBC of rat blood.

Groups SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) GR (mU/mg protein) G6-PDH (U/mg protein)

Control 4.54 ± 0.24 6.23 ± 0.32 1.27 ± 0.42 35.72 ± 1.23 0.29 ± 0.12
H2 O2 3.12 ± 0.19a 5.12 ± 0.17a 0.77 ± 0.23a 21.93 ± 0.78a 0.16 ± 0.09a
H2 O2 + CRE 50 3.91 ± 0.21b 5.77 ± 0.43b 1.12 ± 0.29b 32.01 ± 1.04b 0.23 ± 0.1b

Values are mean ± SD of triplicate determination.

a
P < 0.05 versus control group.
b
P < 0.05 versus H2 O2 treated group.

A B
250 200
*

Time spent in open arm

* *
No of line crossings

200
* 150
*
150
100
*
100

50
50

0 0
Control CRE 100 CRE 200 Diazepam Control CRE 100 CRE 200 Diazepam

C 150
*
120 *
*
Time spent in
light box

90

60

30

0
Control CRE 100 CRE 200 Diazepam

D i 75 D ii
60
* 5
*
4
* *
No of licks

No of shocks

45 3
30 * 2 *
15 1

0 0
Control CRE 100 CRE 200 Diazepam Control CRE 100 CRE 200 Diazepam

Fig. 5. Anxiolytic activity of C. rotundus analyzed by behavioural analysis of mice (A) Openfield test. (B) Elevated plus maze test. (C) Light–dark box test. (D) Thirsty rat conflict
paradigm. The results are represented as mean± SD of three independent experiments. The results were analyzed by one-way ANOVA. Significance was set at * P < 0.05 and
all comparisons were made against the control group (n = 8).

natural or synthetic compounds. This method is based on princi- for oxygen transport, respiration and as a cofactor in the activity
ple that the amount of hydrogen donating antioxidants required of many enzymes. Iron can stimulate lipid peroxidation by Fenton
to reduce DPPH* concentration by 50% in alcoholic solution to reaction, and also accelerates peroxidation by decomposing lipid
form the non-radical form DPPH–H. Our results demonstrate that hydroperoxides into peroxyl and alkoxyl radicals that can them-
C. rotundus extracts dose dependently scavenged the DPPH radi- selves abstract hydrogen and perpetuate the chain reaction of lipid
cals. Iron is an essential element for life and it is mainly required peroxidation. Thus it is an extremely reactive metal and causes the

A 80 B 80
% Inhibition of cell

% LDH released

60 60
viability

40 40

20 20

0 0
10 25 50 100 150 200 400 500 1000 10 25 50 100 150 200 400 500 1000
CRE ( g/ml) CRE ( g/ml)

Fig. 6. (A) Cytotoxicity of C. rotundus by MTT assay. (B) LDH release assay. The results are represented as mean± SD of three independent experiments.
824 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826

oxidative damage to lipids, proteins and other cellular components The diet enriched with antioxidant principles safeguards the cell
(Jomova and Valko, 2011). Therefore, the ability of different extracts against the free radical induced damage. Our observed results are
to chelate/bind metal ion was tested in the present study. The blue in consistence with an earlier report on antioxidant activity of the
green ABTS radical decolouration assay is widely used to assess total oligomeric flavonoid extract of C. rotundus against cerebral
the antioxidant activity of plant extracts. Phenolic compounds have ischaemic-reperfusion injury in rats (Sunil et al., 2011).
been studied as an important contributor to the antioxidant activ- Acetylcholinesterase catalyses the hydrolysis of neurotransmit-
ity of various plant extracts. In an earlier study, Chen and Yen ter acetylcholine and inhibits the termination of neurotransmission
(2007) have investigated the antioxidant activity of different cul- at cholinergic synapses. Acetylcholineesterase inhibitors like
tivars of guava by ABTS radical scavenging activity, and correlated tacrine, donepezil, rivastigmine, and galanthamine are generally
the activity with the polyphenol content. Similarly, our results also used in treating neurodegenerative disorders. Because of the side
demonstrate that the ABTS radical scavenging activity might be effects associated with these drugs, it is worthwhile to explore the
associated with high content of polyphenols present in C. rotun- herbal remedies as an alternative and complementary medicine
dus. The reducing ability of C. rotundus extracts was analyzed by to the existing pharmacological agents. Our observed results are
FRAP assay. This activity depends on the ability of extract to donate in line with an earlier study by Mukherjee et al. (2007) who
hydrogen atoms to inactivate the free radical formation. All the reported the acetylcholineesterase inhibitory activity of various
fractions C. rotundus showed the reducing activity in a dose depen- plant extracts.
dent manner with increase in concentrations. The observed results Anxiety is psychiatric disorder that affects all age groups
are in accordance with a recent report by Ero˘glu Özkan (2013) who of the population in both developed and developing countries.
demonstrated the biological activities of Hypericum pamphylicum. Benzodiazepines that non-selectively target GABAA receptors are
Nitric oxide (NO) or reactive nitrogen species (RNS) have been used as anxiolytes (Rudolph and Knoflach, 2011). Various reports
reported to alter the structural and functional behaviour of many demonstrate the link between anti-oxidant and anti-anxiety activ-
cellular components particularly proteins. In biological system, NO ities (Hovatta et al., 2010; Bouayed, 2011). Glyoxylase-1 and
is synthesized by nitric oxide synthase as a byproduct by conver- glutathione-1 are the genes that are responsible for the anxi-
sion of l-arginine to l-citrulline. The NO-mediated peroxynitrite ety in mice by regulated expression (Hovatta et al., 2005). In
formation results in protein nitration and its effects have also been the present study, we analyzed the anxiolytic effects of C. rotun-
implicated in the pathophysiology of neurodegenerative disorders dus by behavioural tests such as open field test, elevated pluz
(Szabó et al., 2007). The present study indicates that the rhizome maze, light–dark box test and Vogel conflict test. These tests dif-
of C. rotundus is a rich source of antioxidant principles, and there- fer from each other and widely used to assess the behaviour of
fore could play a role in arresting the chain of reactions initiated experimental animals to evaluate the anxiolytic effects of herbal
by excess generation of NO. Our results are in line with the recent extracts/compounds.
report (Konrath et al., 2012) published for nitric oxide scavenging The open field test determines the exploratory activity and we
activities of Lycopodium species. observed a decrease in defecation which is a good indicator of emo-
Several herbal extracts have been reported to protect the radical tionality and its reduction can be associated with anxiolytic effects
mediated DNA damage (Del Bo et al., 2010; Ilaiyaraja and Khanum, of C. rotundus. The elevated plus maze test is based on the observa-
2011). DNA damage has been observed in several stress condi- tion of test animals to display an aversion to open spaces. Increase
tion such as cancer, diabetics and neuronal diseases (Jackson and in time spent in open arms is interpreted as anxiolytic effect and the
Bartek, 2009). Our observed results demonstrate the eukaryotic and same was observed with C. rotundus administration. Anxiety is gen-
prokaryotic DNA damage protective effects of C. rotundus and its erated in the light–dark box test by the conflict between the desire
application to treat DNA damage. to explore and to retreat from an unknown and well-illuminated
Oxidatively modified proteins are highly resistant to proteolytic space (Crawley and Goodwin, 1980). The number of transitions
degradation, and accumulate as inclusion bodies, like amyloid and the time spent in the light chamber increased with C. rotun-
protein fragments in neurodegenerative diseases such as Parkin- dus supplementation which demonstrate the anxiolytic properties.
son’s and Alzheimer’s disease (Butterfield and Kanski, 2001). Our The Vogel conflict test is a rapid screening method to analyze the
results are in consistent with a recent study on protein oxida- anxiolytic properties of test samples. Here, the punished drinking
tion preventing effect of aerial parts of C. rotundus against fructose behaviour with mild electrical shocks leads to reduced water con-
mediated protein glycoxidation by Ardestani and Yazdanparast sumption of deprived animals which was increased in C. rotundus
(2007). Protein nitration has been documented in a variety of group. Our observed results are in corroboration with anxiolytic
disorders and therefore the estimation of 3-NT is a hall mark properties of Echinacea preparation and ␣-asarone demonstrated
to quantify the protein nitration (Szabó et al., 2007). SIN-1 (3- by Haller et al. (2013) and Liu et al. (2012).
morpholinosydnonimine (SIN-1) is a generator of peroxinitrite In order to determine the efficacy of herbal extracts for their pro-
radical, that can be produced by interaction of H2 O2 and NO in cells. tective effect, the LD50 of the extracts should be estimated. Here,
It is also capable of crossing lipid bilayers and causes oxidization, we determined the cytotoxic dose of C. rotundus by MTT and LDH
nitration of tyrosine residues to form nitrotyrosine (Khairutdinov assays. The MTT assay determines cytotoxicity based on the mito-
et al., 2000). Present study demonstrates the protective effect chondrial damage of the cells. In contrast LDH assay determines the
against protein nitration which is in agreement with our recent cytotoxicity based on the plasmamembrane damage of the cells.
report on nitric oxide preventing efficacy of C. rotundus (Hemanth The present study demonstrated the maximum dose of hydroalco-
Kumar et al., 2013). holic fraction of C. rotundus 100 ␮g/mL, to be used for studying the
The free radical generated oxidative stress has been exten- protective effect using SH-SY5Y cells.
sively reported in stress/diseased states as well as in ageing. The
radical mediated stress condition, initiates with accumulation of
O2 •− radicals in cells. SOD enzyme dismutates O2 •− to H2 O and O2 , 6. Conclusion
whereas catalase and GPx scavenge the H2 O2 radicals by converting
it to H2 O and O2 . During oxidative stress, the lipid peroxida- The results of the present study indicates that the hydroalco-
tion products formed by oxidation of polyunsaturated fatty acids holic extract of C. rotundus is effective in preventing the oxidative
induce DNA and cell membrane damage and by generation of DNA stress induced by various radicals viz., DPPH, AAPH, and ABTS, as
adducts which aids in apoptotic damage of cell (Kutuk et al., 2004). evidenced by in vitro free radical scavenging assays. The extract
 K. Hemanth Kumar et al. / Industrial Crops and Products 52 (2014) 815–826 825

showed significant prokaryotic and eukaryotic DNA damage pro- Haller, J., Freund, T.F., Pelczer, K.G., Füredi, J., Krecsak, L., Zámbori, J., 2013. The anx-
tection induced by H2 O2 on plasmid and genomic DNA and also iolytic potential and psychotropic side effects of an echinacea preparation in
laboratory animals and healthy volunteers. Phytother. Res. 27, 54–61.
exhibited a significant protection against protein oxidation and Hemanth Kumar, K., Tamatam, A., Pal, A., Khanum, F., 2013. Neuroprotective effects
nitration induced by AAPH and SIN-1. Moreover C. rotundus also of Cyperus rotundus on SIN-1 induced nitric oxide generation and protein nitra-
showed acetylcholineesterase inhibitory activity and anxiolytic tion: ameliorative effect against apoptosis mediated neuronal cell damage.
Neurotoxicology 34, 150–159.
effects. Therefore C. rotundus can be used as a source of antioxi- Hovatta, I., Juhila, J., Donner, J., 2010. Oxidative stress in anxiety and comorbid
dant for dietary supplementation to alleviate oxidative stress and disorders. Neurosci. Res. 68, 261–275.
anxiety related disorders. Hovatta, I., Tennant, R.S., Helton, R., Marr, R.A., Singer, O., Redwine, J.M., Ellison,
J.A., Schadt, E.E., Verma, I.M., Lockhart, D.J., Barlow, C., 2005. Glyoxalase 1 and
glutathione reductase 1 regulate anxiety in mice. Nature 438, 662–666.
Conflict of interest statement Ilaiyaraja, N., Khanum, F., 2011. Antioxidant potential of Tinospora cordifolia extracts
and their protective effect on oxidation of biomolecules. Phcog. J. 3, 56–62.
Jackson, S.P., Bartek, J., 2009. The DNA-damage response in human biology and
We declare that we have no conflict of interest. disease. Nature 461, 1071–1078.
Jeong, S.J., Miyamoto, T., Inagaki, M., Kim, Y.C., Higuchi, R., 2000. Rotundines A–C,
three novel sesquiterpene alkaloids from Cyperus rotundus. J. Nat. Prod. 63,
Acknowledgments 673–675.
Jomova, K., Valko, M., 2011. Advances in metal-induced oxidative stress and human
disease. Toxicology 283, 65–87.
The authors are highly thankful to Dr. Harshavardhan Batra, Jung, S.H., Kim, S.J., Jun, B.G., Lee, K.T., Hong, S.P., Oh, M.S., Jang, D.S., Choi, J.H., 2013. ␣-
Director, DFRL, Mysore, for his constant encouragement and sup- Cyperone isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced
COX-2 expression and PGE2 production through the negative regulation of NF␬B
port throughout the investigation. The authors would like to
signalling in RAW 264.7 cells. J. Ethnopharmacol. 147, 208–214.
thank Prasanth Bitla, School of Life Sciences, University of Hyder- Khairutdinov, R.F., Coddington, J.W., Hurst, J.K., 2000. Permeation of phospholipid
abad, Hyderabad, for LC–ESI-MS/MS analysis. Kandikattu Hemanth membranes by peroxynitrite. Biochemistry 39, 14238–42491.
Kumar is highly thankful to CSIR for providing SRF. Kilani, S., Ben Ammar, R., Bouhlel, I., Abdelwahed, A., Hayder, N., Mahmoud, A.,
Ghedira, K., Chekir-Ghedira, L., 2005. Investigation of extracts from (Tunisian)
Cyperus rotundus as antimutagens and radical scavengers. Environ. Toxicol. Phar-
macol. 20, 478–484.
Appendix A. Supplementary data Kilani-Jaziri, S., Bhouri, W., Skandrani, I., Limem, I., Chekir-Ghedira, L., Ghedira, K.,
2011. Phytochemical, antimicrobial, antioxidant and antigenotoxic potentials of
Supplementary material related to this article can be found, Cyperus rotundus extracts. S. Afr. Med. J. 77, 767–776.
Kilani-Jaziri, S., Neffati, A., Limem, I., Boubaker, J., Skandrani, I., Sghair, M.B., Bouhlel,
in the online version, at http://dx.doi.org/10.1016/j.indcrop. I., Bhouri, W., Mariotte, A.M., Ghedira, K., Dijoux Franca, M.G., Chekir-Ghedira,
2013.11.040. L., 2009. Relationship correlation of antioxidant and antiproliferative capacity
of Cyperus rotundus products towards K562erythroleukemia cells. Chem. Biol.
Interact. 181, 85–94.
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