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Antioxidative and anti-proliferative potential of Curculigo orchioides Gaertn in oxidative stress induced cytotoxicity: In vitro, ex vivo and in silico studies

  • Centre for Interdisciplinary Research in Basic Sciences
  • Sri Pratap College


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Food and Chemical Toxicology
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Antioxidative and anti-proliferative potential of Curculigo orchioides Gaertn
in oxidative stress induced cytotoxicity: In vitro,ex vivo and in silico studies
Iram Iqbal Hejazi
, Rashmin Khanam
, Syed Hassan Mehdi
, Abdul Roouf Bhat
M.Moshahid Alam Rizvi
, Sonu Chand Thakur
, Fareeda Athar
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, India
Department of Biosciences, Jamia Millia Islamia, India
Sri Pratap College, Cluster University, Srinagar, India
Curculigo orchioides Gaertn
Molecular docking
Plant phytoconstituents have been a valuable source of clinically important anticancer agents. Antioxidant and
anticancerous activity of plant Curculigo orchioides Gaertn were explored In vitro antioxidant activity, anti-
oxidant enzyme activity of oxidatively stressed tissue, and cell culture studies on human cancer cell lines HepG2,
HeLa and MCF-7 were carried out. Active plant fractions were subjected to GC-MS analysis and compounds
selected on the basis of their abundance were screened in silico with the help of Auto Dock 4.2 tools with pre-
selected antioxidant enzymes. Curculigo orchioides Gaertn plant fractions exhibited signicant antioxidant ac-
tivities by virtue of scavenging of free radicals having IC
value of ethylacetate fraction (EA) for DPPH radical
scavenging assay to be 52.93 ± 0.66 μg/ml. Further, antioxidant enzyme defense of mammalian tissue when
treated with plant fractions revealed that enzyme concentrations were refurbished which were increased during
oxidative stress. MTT assay on cell lines HepG2, HeLa and MCF-7 presented IC
values of ethylacetate (EA)
fraction as 171.23 ± 2.1 μg/ml, 144.80 ± 1.08 μg/ml and 153.51 μg/ml and aqueous ethylacetate (AEA)
fraction as 133.44 ± 1.1 μg/ml, 136.50 ± 0.8 μg/ml and 145.09 μg/ml respectively. Further EA and AEA plant
fractions down regulated the levels of antiapoptotic Bcl-2 expression and upregulated the expression of apoptotic
proteins caspase-3 and caspase-8 through an intrinsic ROS-mediated mitochondrial dysfunction pathway.
Key message: Key ndings explained that fractions of Curculigo orchioides Gaertn inhibited oxidative stress by
increasing the antioxidant enzyme content and have anticancerous potential on cancer cell lines HepG2, HeLa
and MCF-7.
1. Introduction
Reactive oxygen species (ROS) act as a second messenger in cell
signaling and are essential for various biological processes in normal
cells. Any aberrance in redox balance may relate to human pathogen-
esis including cancers (Liou and Storz, 2010;Wang and Yi, 2008). ROS
cause oxidative damage to proteins, deoxyribonucleic acid and lipids.
These cytotoxic properties of ROS explain the evolution of complex
arrays of non-enzymatic and enzymatic detoxication mechanisms in
plants. Increasing evidence indicates that ROS also function as signaling
molecules in plants and are implicated in regulating development and
defense responses against pathogens (Chtourou et al., 2016). While
oxidation is the utmost common natural and energy generating reac-
tion, oxidative stress is detrimental to cell, because the oxidation ad-
ducts such as free radicals and reactive oxygen species harm the cellular
machineries, which contribute numerous diseases. Injury to genetic
material is accountable for cancer development and advancement
(Halliwell, 2007b). In aerobic metabolism, production of reactive
oxygen species (ROS) is nearly well-adjusted by means of endogenous
antioxidant defence systems which include enzymes such as superoxide
dismutase, catalase, glutathione peroxidase, glutathione reductase,
glutathione S-transferase (Hejazi et al., 2017;Maciejczyk et al., 2017;
Wang and Yi, 2008). Since the redox balance is not awless, some ROS-
mediated injury happens uninterruptedly. In other words, antioxidant
Received 16 December 2017; Received in revised form 14 February 2018; Accepted 10 March 2018
Corresponding author. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, India.
Current address: Post doctoral fellow Department of Geriatrics Institute of aging University of Arkansas medical sciences ar-little rock-72205 USA.
E-mail addresses: (I.I. Hejazi), (R. Khanam),, (S.H. Mehdi), (A.R. Bhat), (M.M.A. Rizvi), (S.C. Thakur), (F. Athar).
Abbreviations: GC-MS, Gas Chromatography Mass Spectrometry; L-AA, L-ascorbate; SOD, Superoxide Dismutase; CAT, Catalase; GR, Glutathione Reductase; GST, Glutathione S
Transferase; GPx, Glutathione Peroxidase; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; DAPI, 4,6-Diamidino-2-phenylindole dihydrochloride; MTT, 3-(4,5-dimethyl-2-yl)-2,5-diphynyl tetra-
zolium bromide; EA, Ethylacetate; AEA, Aqueous Ethylacetate; AB, Aqueous Butanol
Food and Chemical Toxicology 115 (2018) 244–259
Available online 12 March 2018
0278-6915/ © 2018 Elsevier Ltd. All rights reserved.
defence control levels of ROS rather than eliminate them. So anti-
oxidant defence enzyme system plays a major role in the prevention
from oxygen related damage and further from oncogenesis (Liou and
Storz, 2010;Wang and Yi, 2008).
It is reported that incidence of cancer has been increasing in de-
veloping countries, and has become the fourth leading cause of death
worldwide (Hasan et al., 2016). Chemoprevention by phytoconstituents
has evolved as an eective strategy to control the prevalence of cancer
The quest of anticancer agents from plant sources started its earnest in
the 1950's with the discovery of the vinca alkaloids, vincristine, vin-
blastine, combretastatin and colchicine (Stanton et al., 2011). Epide-
miological studies have established a positive correlation between in-
creased consumption of natural products with decreased risk of cancer
(Murali and Kuttan, 2015). The mechanisms accountable for chemo-
prevention still remains principally unidentied but are probably re-
lated to the presence of phytochemicals associated with plants. There-
fore the search for eective and safer natural anticancer agents has
become an area of current research all over the world.
Curculigo orchioides Gaertn (Hypoxidaceae) (plant name has been
checked with http://www.the and plant has been identi-
ed from the botanical department of Jamia Hamdard, New Delhi.
commonly known as kali musli is a perennial herb with long cylindrical
rhizomes found in subtropical Himalayas to Western Ghats and Konkan
southwards. It is one of the important drugs used for its traditional
aphrodisiac properties (Chauhan et al., 2007). Traditional medicine is
the combination of knowledge, skills, and practices based on the the-
ories, beliefs, and experiences indigenous to dierent cultures used in
the maintenance of health, prevention of diseases, and improvement of
physical and mental illness. The rhizomes, well-known as curculiginis
rhizoma in the Chinese Pharmacopoeia, owns antiosteoporotic, anti-
oxidant, estrogenic, neuroprotective and antibacterial activities. Its
roots are tuberous and rhizomes are slightly bitter and mucilaginous in
taste, and are also used as tonic and demulcent, (Chauhan et al., 2007;
Farzinebrahimi et al., 2016;He et al., 2015;Murali and Kuttan, 2015;
Shanthamma, 2009). The rhizome of the plant also possess numerous
other medicinal properties such as, diuretic, aphrodisiac, antiasthmatic,
antibronchitis and antijaundice (Ramchandani et al., 2014;
Shanthamma, 2009). The interesting immense medicinal importance
encouraged us to further explore the phytochemical investigation on
Curculigo orchioides Gaertn. Since antioxidant potential of plants are
directly linked to their anticancerous potential (Dai and Mumper, 2010;
Elsharkawy, 2017), we therefore, aimed to nd out antioxidative and
anticancerous potential of Curculigo orchioides Gaertn species in cell free
system, cell system and animal system.
2. Materials and methods
2.1. Procurement of plant material
Dried rhizomes of Curculigo orchiodes Gaertn were collected from
local market of Delhi. The authenticity of the rootstocks was established
from Department of Botany, Jamia Hamdard, New Delhi and the vou-
cher specimen was deposited in the University Centre.
2.2. Chemicals and reagents studied
The key chemical compounds studied in this article are L-Ascorbic
acid (PubChem CID: 54670067), L-nitro-arginine methyl ester (L-
NAME), 2,2-diphenyl-1-picrylhydrazyl (DPPH), (3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), 2,4-dini-
trophenylhydrazine (DNPH), Griess reagent, trichloroacetic acid, anti-
biotic solution, fetal bovine serum sodium nitro prusside, ferric
chloride, hypochlorous acid, reduced glutathione, oxidized glutathione,
tetra methoxy propane, dimethyl sulphoxide (DMSO), glutathione re-
ductase, hydrogen peroxide, capsaicin (PubChem CID: 1548943), pi-
perine (PubChem CID: 638024).
2.3. Plant extraction
The hot sohxlet extraction method using organic solvents was im-
plemented to acquire extracts from the rhizomes of the plant Curculigo
orchioides Gaertn (Sultana et al., 2009).
2.4. Determination of phenolic, avonoid and proanthrocyanidin content
The total phenolic avonoid and proanthrocyanidin content of the
crude extracts was determined according to the previously studied
methods(Dai and Mumper, 2010;Sahu et al., 2017). After performing
the initial phytochemical screening, plant fractions were further eval-
uated for antioxidant activity using various in vitro assays. In the ex-
periments, rutin was taken as standard for total avonoid content,
gallic acid was taken as reference/standard antioxidant for total phe-
nolic content and catechin was taken as reference for proan-
throcyanidin content.
2.5. Determination of in vitro antioxidant activity
The DPPH radical scavenging was done according to the previously
studied methods with slight modications (Camilo López-Alarcóna,
2013;Tacchini et al., 2015). Nitric oxide scavenging activity was esti-
mated using Griess reaction (Chung et al., 2017;Hassan et al., 2017;
Márcio Carocho, 2013). Total reduction capability was also done to
ensure the reduction capability of the plant according to previously
studied methods (Mladenov et al., 2015;Tacchini et al., 2015). Su-
peroxide dismutase scavenging activity, hypochlorous acid scavenging
activity and total antioxidant activity were conducted according to
Sharma et al. (2014).
2.6. Ex vivo antioxidant enzyme assays
2.6.1. Preparation of tissue homogenate
The research was conducted in accordance with the internationally
accepted principles for the laboratory animal use and care. We thank
the Director of slaughter house, Ghazipur, Delhi, for organizing goat
liver tissues. The tissues were washed in normal saline and lipid layer
was removed. 1 g of liver tissue was weighed and very thin slices were
made from them (89 mm rectangular shape). Further washing was
done in PBS buer (pH 7.2). The ex vivo model was then confronted
with the oxidant H
, in presence and absence of various concentra-
tions of extracts. The components were analyzed spectro-
photometrically in homogenate after one hour of incubation at 37 °C
(Meera et al., 2009).
2.6.2. Estimation of lipid peroxidation and protein oxidation
Free radicals produce lipid peroxidation mechanism in cells.
Malondialdehyde (MDA) is one of the chief yields of fatty acids per-
oxidation in the cells. An upsurge in free radicals grounds for the pro-
duction of MDA. Malondialdehyde levels are usually recognized as
markers of oxidative stress and the antioxidant status in cancerous
patients. Thiobarbitoric acid (TBA) assay is the most frequently used
method for determining MDA in biological solutions. The assay is based
on a condensation reaction of two molecules of TBA with one molecule
of MDA. MDA reacts with Thiobarbitoric acid (TBA) to produce a pink
colour which is read at 532 nm spectrophotometrically (Belardinelli
et al., 2007;Kim et al., 2017). Protein oxidation was estimated by
measuring the protein carbonyl level by the earlier described methods
(Jyoti et al., 2009;Sahu et al., 2017).
2.6.3. Estimation of SOD, CAT, GR, GPx and GST
Superoxide dismutase (SOD) and catalase (CAT) assays were per-
formed according to previous studied methods with slight modications
(Sharma et al., 2014). Glutathione Reductase (GR), Glutathione-S-
transferase (GST), Glutathione peroxidase (GPx) were also measured
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
according to previous methods (Hejazi et al., 2017;Pahwa et al., 2017;
Sahu et al., 2017;Weydert and Cullen, 2010).
2.7. Cytotoxicity studies on cancer cell lines HepG2, HeLa and MCF-7
2.7.1. Cellular experiments
Cell lines HepG2, HeLa and MCF-7 were obtained from National
Centre for Cell Sciences (Pune). Cells were fully grown in Dulbecco's
Modied Eagle's Medium accompanied with 10% Fetal Bovine Serum,
1% antibiotic solution encompassing penicillin, streptomycin and am-
photericin B, and 10 mM of HEPES and 25 mM of Na
in a dam-
pened atmosphere of 5% CO
at 37 °C in air jacketed incubator. Stock
culture was sustained in the exponential growth phase by subculturing
as monolayer culture using 0.02% EDTA. The dislodged and extricated
cells were suspended in whole medium and reseeded regularly (Hejazi
et al., 2017;Mallick et al., 2016).
2.7.2. Cytotoxicity assay/MTT assay
The cytotoxic eect was evaluated in HepG2, HeLa and MCF-7
cancer cell lines exposed to various concentrations of EA extract and
AEA extract of Curculigo orchiodes Gaertn by the MTT assay. Cancer cell
lines were procured from National Centre for Cell Sciences (Pune). Cells
were seeded over night at the number of 1 × 10
per well and then
nurtured with a number of concentrations of extracts ranging from 50
to 300 μg/ml for 48 h respectively. At the end of the treatment, medium
was cast-oand cells were treated with 20 μl of MTT (5 mg/ml in PBS)
in fresh medium (50 μl) for 4 h in CO2 incubator. After four hours
formazan crystals were formed due to mitochondrial reduction of MTT.
Those were dissolved in DMSO (150μl/well) and the absorbance was
read at 570 nm(Hejazi et al., 2017;Mallick et al., 2016).
2.7.3. DAPI staining to visualize apoptosis: uorescent microscopy
4, 6-diamidino-2-phenylindole or commonly known as DAPI is a
ourescent dye that xes sturdily to A-T rich regions in DNA. To assess
apoptosis consequently after drug treatment, cells were marked uor-
escently with (DAPI) to sense nuclear condensation and fragmentation.
Concisely, HepG2, HeLa and MCF-7 cells (1 × 10
) seeded in12-well
plate were treated with Thymol and Palmitic acid. After that, cells were
washed thrice in phosphate-buered saline (PBS) and immobilized with
250 μL paraformaldehyde (4%) solution at 25 °C for about 8 min. These
xed cells were then washed with PBS and permeabilized with 0.1%
Triton-X 100 for 10 min and again washed with PBS thrice. Finally, cells
were stained with a DAPI solution (2 μg/ml) (Himedia) at room tem-
perature in the dark. The nuclear morphology of the cells was scruti-
nized by uorescent microscopy(Hejazi et al., 2017;Khanam et al.,
2.7.4. Western blot analysis
After the completion of transfection experiments in dierent cell
lines as described above, cells were lysed with lysis buer (Tris 50 mM,
pH7.4, NaCl 250 mM, EDTA 5 mM, 0.5% TritonX 100, Na
VO4 1 mM)
containing protease inhibitor cocktail. Followed by complete lysis, the
cells were centrifuged at 12000g for 15 min to obtain the clear cell
lysate. Protein concentration was determined by using Standard
Bradford assay. 40 μg of lysate was diluted with 6X Laemmli's buer,
boiled for 35 min and were set on 12% SDS-polyacrylamide gel under
reducing conditions. Further the resultant polypeptides were electro-
phoretically transferred to nitrocellulose membrane in presence of 5%
BSA to block the nonspecic antibodies. The membrane was incubated
overnight at 4 °C with the explicit primary antibodies followed by
horseradish peroxidase (HRP) conjugates and then perceived by H
and DAB in the ratio of 1:1 (Hejazi et al., 2017;Khanam et al., 2018;
Mallick et al., 2016).
2.8. GC-MS analysis of the plants active extracts
GC-MS analysis of the most active fractions ethylacetate (EA),
aqueous ethylacetate (AEA) plant fractions was done. The crude ex-
tracts were analyzed by GC-MS using a Thermonnigan Trace GC-MS
single quadrupole mass spectrometer with AS 800 auto sampler. The
separations were accomplished by capillary column, Phenomenex ZB5
(30 m × 0.25 mm, lm thickness 0.25 μm). The column temperature
was kept at 40 °C for 4 min and then at dierent temperatures (160 °C,
220 °C) at variable rates (10 deg/min, 2 deg/min) for 10 min. The ow
rate of helium as carrier gas was 1 ml/min. MS was taken at 70eV
electron ionization trap current 150 μA and source temperature
200 °C(Hejazi et al., 2017).
2.9. Molecular docking studies
Compound from the plant fractions, as reported in the GC-MS
analysis were selected based on their drug like properties, their non-
violation of Lipinski rule of 5 and ADMET properties determination by
Discovery Studio from Accelerys. The crystal structure of proteins de-
termined at a resolution 2.5 Å, were retrieved from the protein data
bank as docking templates. Four antioxidant enzymes (proteins) were
downloaded from protein data bank with their PDBID s as superoxide
dismutase (4mcm), catalase (1dgb), glutathione peroxidase (3kij), and
glutathione-S-transferase (4mpg). To investigate the ability of anti-
oxidant agents, the molecular docking was rst conducted with L-
Ascorbate (strong antioxidant, which is used as reference ligand). The
molecular docking studies have been carried out according to the
method of Hejazi et al. (2017) and Prakash Amresh et al. (2013).
3. Results and discussions
3.1. Plant extraction
The extraction procedure we followed was continuous hot solvent
extraction which involved the separation of medicinally active portions
or secondary metabolites of plant tissues from the inert components by
using selective solvents. The advantage of this method, is that enormous
amount of drug can be extracted with a trivial quantity of solvent
(Sultana et al., 2009). During extraction, solvents diuse into the solid
plant material and solubilize compounds with similar polarity. Extracts
of the plant in various solvents were obtained using polarity index of
the solvents in their ascending order. We obtained eight fractions.
Table 1 shows yield of the plant fractions in various solvents.
3.2. Phytochemical screening and total phenolic, avonoid and
proanthocyanidin content
Polyphenols are secondary metabolites of plants and are frequently
involved in protection against reactive oxygen species. Studies have
supported a strong association amidst antioxidant activity of plant ex-
tracts and their phenolic content, possibly due to their redox features,
which licenses them to perform as reducing agents, free radical sca-
vengers and hydrogen donors(Castro-Concha et al., 2014).
In the past span of time, there has been much attention in the
prospective health benets of dietary plant polyphenols as antioxidants.
Epidemiological studies and associated meta-analyses strongly propose
that long term intake of diets rich in plant polyphenols oer defense
against development of cancers (Garcia-Calzon et al., 2015;Kaulmann
et al., 2014). Here we oered acquaintance about the biological eects
of plant polyphenols in the context of signicance to human wellbeing
and health. The phenolic (176.58 ± 0.160 mg gallic acid equivalents
per gram of extract) and avonoid (94.61 ± 0.089 mg rutin equiva-
lents per gram of extract) contents of plant's EA fraction were markedly
higher than that of the proanthocyanidin (86.61 ± 0.77 mg catechin
equivalents per gram of extract) content. Results are shown in Table 1.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
3.3. In vitro antioxidant assays
3.3.1. DPPH free radical scavenging activity
The free radical scavenging activity of C. orchioides fractions on
DPPH radicals was calculated by determining the competency of anti-
oxidants present in the fractions to reduce the stable DPPH radical
(purple) to its non-radical form DPPH (yellow) (Kalaycioglu and Erim,
2017). Although EA fraction was not found to be as ecient as L As-
corbate, but its activity was highest amongst all the other fractions. This
can be due to the fact that hydrogen-donating compounds are more
expected to be present in polar solvents. Results presented in Fig. 1a
shows that plant fractions inhibited DPPH radicals in a dose-dependent
manner with an IC
value of EA fraction (52.93 ± 0.66 μg/ml) fol-
lowed by ether fraction (57.04 ± 0.98 μg/ml), AEA fraction
(68.35 ± 0.76 μg/ml) and AB fraction (69.81 ± 0.36 μg/ml). L-AA
showed a lower value (39.3 ± 0.43 μg/ml). At the concentration of
800 μg/ml, scavenging abilities of all plant fractions on DPPH radicals
expressed as %inhibition (p > 0.005) were non-signicant as com-
pared to that of L-AA.
3.3.2. Nitric oxide radical scavenging
C orchioides markedly condensed the extent of nitrite produced by
the decomposition of sodium nitroprusside and was found to be equally
eective with L-ascorbate. Fig. 1b shows that plant fractions exhibited a
moderate dose-dependent nitric oxide scavenging activity between 5
and 800 μg/ml. IC
values of AEA fraction (65.003 ± 0.67 μg/ml)was
the lowest after standard compound L-AA (38.9 ± 0.32 μg/ml) fol-
lowed by AB fraction (82.90 ± 0.98 μg/ml) and EA fraction
(141.22 ± 0.44 μg/ml) respectively. The potency in terms of percen-
tage inhibition (p > 0.005) was statistically insignicant as compared
to the values of standard compound L-AA and is given as; L-AA
(87.77 ± 2.24)% > AEA fraction (90.10 ± 0.18)% > EA fraction
(89.96 ± 0.16)% > AB fraction (88.2 ± 1.65)% > Chloroform
fraction (88.04 ± 1.42)% > Aq. fraction (85.69 ± 2.29)% >
Hexane fraction (85.09 ± 4.89)% > Methanol fraction (80.96 ±
04)% > Ether fraction (81.23 ± 0.20)%. Moreover, the observed ex-
perimental reduction of nitric oxide might be owing to the antioxidant
properties of C. orchioides, which compete with oxygen to interact with
nitric oxide thus constraining the generation of peroxynitrite. The re-
sults from in vitro studies demonstrated that C. orchioides is a vibrant
scavenger of free radicals at diverse scales of eectiveness. This perhaps
might be due to the phenolic content, principally avonoids existing in
plant, which are hypothetically strong radical scavengers (Adegbola
et al., 2017;Asha et al., 2017;Halliwell, 2007a). Moreover, C. orch-
ioides distinctly compressed the extent of nitrite produced by the de-
composition of sodium nitroprusside and was found to be equally po-
tent as L-ascorbate. The observed experimental reduction of nitric oxide
might be owing to the antioxidant properties of C. orchioides, which
compete with oxygen to interact with nitric oxide thus constraining the
generation of peroxynitrite (Aliancy et al., 2017;Hassan et al., 2017;
Hejazi et al., 2017).3.3.3. Superoxide radical scavenging.
C.orchioides Gaertn signicantly inhibited superoxide radicals in a
dose dependent manner (5800 μg/ml) and the results are shown in
Fig. 1c. At the highest concentration of 800 μg/ml (p > 0.005), EA
fraction exhibited 91.96 ± 0.16% scavenging activity even higher than
standard compound L-AA (88.77 ± 022)% followed by AEA fraction
(87.10 ± 0.18)% and AB fraction (80.20 ± 1.65)%.Signicant dif-
ferences (p < 0.0001) were also observed among the IC
values and
the order was found to be L-AA (37.03 ± 0.22 μg/ml) > EA fraction
(64.10 ± 0.24 μg/ml) > Aq fraction (64.26 ± 0.78 μg/ml) >
AB fraction (66.93 ± 0.55 μg/ml) > Methanol fraction (70.42 ±
0.22 μg/ml) > AEA fraction (75.52 ± 0.89 μg/ml) > Ether fraction
(130.24 ± 0.32 μg/ml) > Chloroform fraction (131.02 ± 0.18 μg/
ml) > Hexane fraction (165.28 ± 0.13 μg/ml).
3.3.3. Hydrogen peroxide radical scavenging
High hydrogen peroxide radical scavenging activities of C. orchioides
Gaertn fractions and the standards were observed at relatively lower
concentrations (Fig. 1d). At 800μg/ml. concentration, L-AA
(91.77 ± 3.04)% EA fraction (88.96 ± 4.16) %, AEA fraction
(91.10 ± 4.18)% and AB fraction (88.20 ± 4.65)% showed almost
equal hydrogen peroxide radical scavenging activity (p > 0.05).
Chloroform fraction (68.04 ± 3.04)% showed the lowest activity. With
respect to IC
values (p < 0.0001), the scavenging eect followed the
order: L-AA (59.76 ± 0.7 μg/ml) > Ether fraction (86.01 ± 0.88 μg/
ml) AEA fraction (90.11 ± 0.52 μg/ml) > AB fraction
(99.0 ± 0.88 μg/ml) > EA fraction (100.76 ± 0.33 μg/ml) >
Methanol fraction (103.99 ± 0.41 μg/ml) > Aq. fraction
(147.79 ± 0.77 μg/ml) > Chloroform fraction (129.19 ± 0.5 μg/
ml) > Hexane fraction (138.52 ± 0.89 μg/ml). Table 2 shows the IC
values of various assays. The results from in vitro studies exhibited that
C. orchioides is an active scavenger of both superoxide and hydrogen
peroxide radicals at dierent scales of potency. This possibly might be
due to the phenolic content, predominantly avonoids present in plant,
which are supposed to be strong superoxide radical scavengers.
3.3.4. Total reduction capability
The reducing power of C. orchioides ethyl acetate fraction expressed
the highest reducing capability as absorbance values increased from
0.195 ± 0.023 to 2.1 ± 0.02 as compared to reducing capabilities of
L-AA which increased from 0.098 ± 0.023 to 1.27 ± 0.22. At 800μg/
ml. reductive capability of EA fraction was higher than that of standard
compound L-AA (Fig. 1e). showed the reducing capability of various
fractions of C. orchioides Gaertn. Hexane showed lowest activity at every
concentration used. No signicant dierence (p > 0.05) was observed
between the reducing power of L-AA, EA fraction, AEA fraction and AB
Table 1
Total extractable components (EC), total phenolic content, total avonoid content proanthrocyanidin content in Hexane, Ether, Chloroform, Ethyl acetate (EA), Methanol, Aq. ethyl
acetate (AEA) and Aq. butanol (AB) extract fractions from Curculigo orchiodes Gaertn extract.
S. No Extracts % EC Total Flavonoid content
Total Phenolic content
Proanthrocyanidin content
1 Hexane 6.9 32.0 5 ± 0.081 108.12 ± 0.494 39.0 5 ± 0.12
2 Ether 3.4 35.88 ± 0.041 125.50 ± 0.145 45.88 ± 0.56
3 Chloroform 6.7 34.05 ± 0.03 42.24 ± 0 .020 42.05 ± 0.63
4 EA 7.3 94.61 ± 0.089 176.58 ± 0.160 86.61 ± 0.77
5 Methanol 8.8 36.05 ± 0.051 69.33 ± 0.063 46.05 ± 0.55
6 Aqueous 10.8 25.88 ± 0.013 83.95 ± 0.025 15.88 ± 0.35
7 AEA 3.2 80.79 ± 0.004 142.29 ± 0.277 61.79 ± 0.43
8 AB 2.9 66.53 ± 0.006 123.20 ± 0.195 56.53 ± 0.63
Values are the mean ± S.D. of three replicates.
Expressed as mg rutin equivalents/g dry weight of plant fraction.
Expressed as mg gallic acid equivalents/g dry weight of plant fraction.
Expressed as mg catechin equivalents/g dry weight of plant fraction.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
3.3.5. Total antioxidant capacity
Fig. 1f shows that at relatively lower concentrations of 550 μg/ml,
EA fraction, AEA fraction and L-AA exhibited almost similar total an-
tioxidant capacity (p > 0.05) whereas at the higher concentrations of
400 and 800 μg/ml, the activity of L-AA and EA was found to be sta-
tistically non-signicant (p > 0.05). At 800 μg/ml this assay exhibited
the following sequence of total antioxidant capacity (p < 0.001) in
terms of optical density: L-AA (3.27 ± 022) > EA fraction
(2.6 ± 0.02) > AEA fraction (2.5 ± 048) > AB fraction
(1.79 ± 0.02) > Methanol fraction (1.05 ± 0.01) > Chloroform
fraction (1.04 ± 0.08) > Ether fraction (1.28 ± 0.12) > Hexane
fraction (0.63 ± 0.02). The reducing power of a plant extract may well
Fig. 1. Eect of variable concentrations of plant fractions and standard antioxidant, L-Ascorbate on inhibition of (a)DPPH radicals, (b)Nitric oxide radicals, (c)Superoxide Radical
Scavenging, (d)Hydrogen peroxide radical scavenging, (e)Total reduction capability, (f)Total antioxidant activity{All the assays were performed in triplicate and the results expressed as
mean standard deviation (SD).
Table 2
of in vitro antioxidant assays.
Plant Fractions Radical Scavenging Assays
DPPH NO Superoxide H
μg/ml IC
μg/ml IC
μg/ml IC
L-Ascorbate 39.3 ± 0.43 38.9 ± .0.32 37.03 ± 0.22 59.76 ± 0.7
Hexane 169.80 ± 0.54 275.09 ± 0.88 165.28 ± 0.13 138.52 ± 0.89
Ether 57.04 ± 0.98 209.75 ± 0.12 130.24 ± 0.32 86.01 ± 0.88
Chloroform 65.24 ± 0.53 314.98 ± 0.8 131.02 ± 0.18 129.19 ± 0.5
EA 52.93 ± 0.66 141.22 ± 0.44 64.10 ± 0.24 100.76 ± 0.33
Methanol 113.35 ± 0.44 323.98 ± 0.32 70.42 ± 0.22 103.99 ± 0.41
Aqueous 104.85 ± 0.66 209.87 ± 0.9 64.26 ± 0.78 147.79 ± 0.77
AEA 68.35 ± 0.76 65.03 ± 0.67 75.52 ± 0.89 90.11 ± 0.52
AB 69.81 ± 0.36 82.90 ± 0.98 66.93 ± 0.55 99.0 ± 0.88
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
Fig. 2. Eects of EA (ethylacetate) and AEA (Aq.ethylacetate) fractions of Curculigo orchioides Gaertn along with standard antioxidant L-Ascorbate treatment on the levels of dierent
enzymatic antioxidants measured in goat liver tissues. Four bars of similar colours represent the concentrations used (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml) on X-Axis. (a) lipid
peroxidation in n moles of MDA/mg protein. (b) protein carbonyl content in units/mg protein Results of (cg) antioxidant enzymes are expressed in units/mg protein. Each value
represents the mean ± S. D of triplicates. p* < 0.05, ⁄⁄, p** < 0.01,⁄⁄⁄p*** < 0.001,///p**** < 0.0001. (For interpretation of the references to colour in this gure legend, the reader
is referred to the Web version of this article.)
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
assist as a signicant indicator of its probable antioxidant activity. The
concentration-dependent, high reducing power of C. orchioides reects
its capability to reduce the transition state of iron and subsequently, the
extent at which superoxide and hydroperoxyl radicals are generated.
3.4. Antioxidant enzyme assays using liver tissue
The activities of all the enzymatic antioxidants in the liver slices
decreased considerably on exposure to H2O2 while treatment with
plant fractions signicantly preserved antioxidant activities.
3.4.1. Lipid peroxidation and protein oxidation
Lipid peroxidation is accountable for the deterioration of membrane
uidity and exibility, inactivation of membrane-bound proteins pre-
dictably leading to the accumulation of cytotoxic aldehydes such as
malondialdehyde (MDA) or hydroxynonenal while the oxidative injury
to proteins interrupts the cellular network and roles of receptors, signal
transduction mechanisms, enzymes and various transports. The changes
in the levels of MDA and protein carbonyl content due to the eect of
oxidative stress are shown in Fig. 2a-b respectively. The H
tissue showed increased lipid peroxidative (4.9-fold, ****p < 0.0001)
and protein oxidative (4.7-fold, ****p < 0.0001) damage when
compared with control tissue.
Lipid peroxidation was decreased in a dose dependent manner when
the liver tissue was treated with plant fractions of C. orchioides extract.
Earlier studies have shown that antioxidative phenols and phenolic
glycosides are present in Curculigo orchioides which are solely re-
sponsible for the various active roles of the plant (Wu et al., 2005).
Treatment of AE fraction brought about 2.9-fold (***p < 0.001)
increase in the stressed tissue, while treatment of AEA brought about
3.6-fold (***p < 0.001) increase in the stressed liver tissue. When we
analyzed protein oxidation the results suggested that protein carbonyl
content in stressed tissues has decreased (***p < 0.001) when treated
with plant fractions in a dose dependent manner. The order of inhibi-
tion of lipid peroxidation and protein oxidation showed by phytocon-
stituents follow as L-AA > AEA > AE.
3.4.2. Status of tissue antioxidant enzymes (CAT, SOD, GPx, GR, and GST)
The antioxidant enzymes (SOD), (CAT), (GPx), (GST) and (GR)
which are biomarkers of OS activities (Hassan et al., 2017) were de-
termined in liver tissue in oxidatively stressed conditions. Results are
shown in Table 4. Experimental results showed that supplementation
with AE and AEA fraction treatment regimen signicantly lowered the
enzymatic (SOD, CAT, GR, GPx and GST) antioxidant status with a
corresponding decrease in the extent of lipid peroxidation markers.
Results are shown in Fig. 2 (cg).
Superoxide dismutase is an enzyme that consecutively catalyzes the
dismutation of the superoxide (O
) radical into ordinary molecular
oxygen (O
) or hydrogen peroxide (H
). Superoxide is created as a
by-product of oxygen metabolism and, if not delimited, causes many
types of cell damage. The protective eect of plant fractions on oxi-
dative stress induced toxicity on antioxidant armory is shown in
Table 3.
Catalase is a common enzyme found in almost all living creatures
exposed to oxygen. It catalyzes the decomposition of hydrogen peroxide
to water and oxygen. It is the chief enzyme in protecting the cell from
oxidative damage by reactive oxygen species (ROS). CAT activity was
markedly increased (***p < 0.001) due to H
administration in
group II as compared to group I. Its activity was restored signicantly
(***p < 0.001) by all the doses except 50 μg/ml of EA treatment
(p > 0.05).
The activity of antioxidant enzymes (SOD), was signicantly ele-
vated (**p < 0.01) in group II as compared to group I. AE fraction
signicantly (***p < 0.001) decreased the activity of enzymes in
group V as compared to group II. The higher dose of 200 μg/ml of AEA
fraction also signicantly restored (****p < 0.0001) SOD activity in
group VI as compared to group II.
The glutathione family comprises of many isozymes with distinct
subcellular sites and display diverse tissue-specic expression patterns.
Currently, the purpose of these enzymes in plants is still vague (Ghosh
et al., 2006;Guevara-Flores et al., 2017). Glutathione S-transferases
(GSTs), are phase II metabolic enzymes known for their capability to
catalyze the conjugation of the reduced form of glutathione (GSH) to
xenobiotic substrates for detoxication (Habig et al., 1974;Pahwa
et al., 2017). GST was augmented in group II (p < 0.001) in oxidative
stress as compared to group I. However all the doses of EA restored GST
signicantly (**p < 0.01). AEA showed no signicant dierence
(p > 0.05) in the activity of GST as compared to group II.
Glutathione peroxidase (GPx) is an intracellular antioxidant enzyme
that enzymatically reduces hydrogen peroxide to water to limit its
harmful eects (Abarikwu et al., 2017;Gokce Cokal et al., 2017). There
was also concomitant and signicant increase in the activity of glu-
tathione peroxidase as compared to control group (II) (***p < 0.001
and **p < 0.01). However, prophylactic treatment of EA at all the
doses replenished the activity of the above said enzymes considerably
(***p < 0.001). Similar results were obtained with the treatment of
AEA at all the doses with statistically signicant dierences between
the stressed conditions and the refurbished states (**p < 0.01). All
results were comparable to standard antioxidants L-AA used as re-
ference compound.
Glutathione reductase (GR) catalyzes the NADPH-driven reduction
of GSSG to GSH. The overall impression that accumulation of disuldes
must be badfor cells further powered the ready recognition of
thoughts that thiol S-thiolation reactions were critical to oxidant-
mediated cell killing (Couto et al., 2016;Kim et al., 2016). GST activity
was markedly increased (***p < 0.001) due to H
administration in
Table 3
Enzyme activity.
Enzyme Activity (200μg/ml) (units/mg protein)
Group I(Blank) 1.32 ± 0.07 07.6 ± 0.08 09.132 ± 0.05 09.56 ± 0.04 07.56 ± 0.48 09.49 ± 0.01
Group II(Blank + H
) 06.21 ± 0.02 10.08 ± 0.06 13.31 ± 0.006 12.05 ± 0.01 08.15 ± 0.12 11.68 ± 0.06
Group III(Blank + H
+L-AA) 01.95 ± 0.005 08.02 ± 0.02 06.14 ± 0.02 08.98 ± 0.018 05.09 ± 0.81 07.43 ± 0.67
GroupIV(Blank + H
+EA) 01.02 ± 0.08 08.06 ± 0.01 09.57 ± 0.16 07.03 ± 0.01 06.31 ± 0.01 06.85 ± 0.16
Group V
(Blank + H
01.41 ± 0.01 06.03 ± 0.02 08.19 ± 0.12 06.33 ± 0.01 05.03 ± 0.011 07.06 ± 0.01
Table 4
Correlation with LPO of EA fraction with antioxidant enzymes.
Enzymes r 95% condence interval R square P (two-tailed)
CAT 0.9071 0.6863 to 0.9985 0.9545 0.0042
SOD 0.9071 0.6863 to 0.9985 0.9545 0.0042
GR 0.9071 0.6863 to 0.9985 0.9545 0.0042
GST 0.9071 0.6863 to 0.9985 0.9545 0.0042
GPX 0.9071 0.6863 to 0.9985 0.9545 0.0042
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
group II as compared to group I. Its activity was restored signicantly
(***p < 0.001) by all the doses except 50 μg/ml of EA treatment
(p > 0.05).
However several previous studies have also reported the nding in
which plant products have been considered as enhancers of antioxidant
enzymes. Al-Rubaei et al. demonstrated that Curcumin has been found
to possess tremendous therapeutic potency as antiinammatory, and
antioxidant agent, and has protective eect from oxidative stress and
enhance total antioxidant capacity in liver damage (Al-Rubaei et al.,
2014). Camileo Lopez focused on their ndings that a diet rich in an-
tioxidants from natural sources can be helpful to human health (Camilo
López-Alarcóna, 2013). Also Kaulman et al. discovered the antioxidant
activity of carotenoids and polyphenols from Brassica oloraceae
(Kaulmann et al., 2014)3.4.3 . Correlation Analysis.
We calculated the correlation factor (Pearson Anderson Coecient)
between lipid peroxidation and all antioxidant enzymes. The results are
shown in Fig. 3ac and Table 4.
3.5. Cellular experiments
3.5.1. MTT cytotoxicity assay
Cancer cell lines HepG2, HeLa and MCF-7 when treated with plant
EA and AEA fractions showed a considerable amount of inhibition of
cells. Apoptosis has occurred due to the strong antioxidant nature of the
fractions which increase the antioxidant enzymes thus helping in the
killing of cancer cells (Fulda and Debatin, 2006a,b). Further studies
need to be done to elucidate the pathways involved. MTT assay on cell
lines HepG2, HeLa and MCF-7 presented IC50 values of EA fraction as
171.23 ± 2.1 μg/ml, 144.80 ± 1.08 μg/ml and 153.51 μg/ml andAEA
fraction as 133.44 ± 1.1 μg/ml 136.50 ± 0.8 μg/ml and 145.09 μg/
ml respectively. Fig. 4a-h showed the percentage inhibition on various
cell lines.
3.5.2. DAPI uorescent staining
Cell death was established through the uorescence microscopic
study. Results indicated that the treated cells (IC
) for 48 h showed the
transformed nuclear morphology. Control cells showed regular mor-
phology, however, in treated cells nuclear morphology seems to have
undergone nuclear condensation, nuclear blebbing, nuclear fragmen-
tation and overall morphological changes are depicted in Fig. 5a-l.
3.5.3. Western blotting
Apoptosis is principally accomplished by a family of proteases
known as the caspases (cysteinyl, aspartate-specic proteases).
Throughout apoptosis, the activation of caspases-3,-8 is regarded as one
of the most noticeable appearances in many cell types (Boege et al.,
2017;Cui et al., 2017). The activation of caspases-3, -8, were assayed
by western blot analysis. As shown in Fig. 6a-d, after the treatment of
cells with IC
concentrations of plant fractions, the levels of caspases-
3, -8, expression increased in all 3 given cell lines (HepG2, HeLa and
MCF-7). Anti-apoptotic Bcl-2 protein has also been strongly trailed as
therapeutic target for apoptosis-inducing anti-cancer approaches due to
evidence of their overexpression in many cancers (Han et al., 2017;
Maji et al., 2018). It was found that the expression of Bcl-2 protein in all
the given cell lines was downregulated after the treatment of cells with
plant AE and AEA fractions.
So we concluded from the western blot studies that HepG2, HeLa
and MCF-7 cells exposed to IC
values of the plant fractions, showed
that the levels of the antiapoptotic protein Bcl-2 expression was down-
regulated, whereas the expression of caspases-3,and-8 were up-regu-
lated. These data show that our plant fractions from Curculigo orchioides
Gaertn can activate caspases specically caspase 3 and caspase 8
Fig. 3. Correlation analysis depict a strong positive association between LPO and various Enzyme Assays (a) Lipid peroxidation assay with all antioxidant enzymes (SOD,CAT, GR,
GST,GPx) treated with standard L-Ascorbate (200 μg/ml) (b) Lipid peroxidation assay with all antioxidant enzymes treated with EA fraction (200 μg/ml) (c) Lipid peroxidation assay with
all antioxidant enzymes treated with AEA fraction (200 μg/ml).
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
(which are considered to be anticancerous proteins) and suppress the
expression of Bcl-2 (which is considered to be cancerous protein) family
3.6. GC-MS analysis
GC-MS analysis of the most active fractions (based on their results of
in vitro antioxidant assays) i.e. EA, AEA of the plant extracts was done
to have a complete proling of the plants compounds. The total ion
chromatogram (TIC) showing the GC-MS prole of the compounds
identied shows that the peaks in the chromatogram were integrated
and were paralleled with the database of spectrum of known compo-
nents deposited in the GC-MS library. The gas chromatogram shows the
relative concentrations of various compounds getting eluted as a
function of retention time. The heights of the peak specify the
comparative concentrations of the components present in the plant. The
mass spectrometer analyzes the compounds eluted at dierent times to
identify the nature and structure of the compounds. The large com-
pound fragments into small compounds giving rise to appearance of
peaks at dierent m/z ratios.
The results of the GCMS analysis of the EA and AEA fractions
(Tables 1 and 2 supplementary les) showed the presence of several
compounds that include heterocyclic compounds, acids, esters etc.
Fig. 1ab in supplementary les show the respective chromatograms of
the EA and AEA fractions.
3.7. In silico ADMET prediction
Pharmokinetic properties and toxicity of the selected compounds
from GC-MS analysis were predicted using Discovery studio from
Fig. 4. Eects of EA and AEA fractions of Curculigo orchioides Gaertn on human cancer cell lines CHO,HeLa, HepG2 and MCF-7 using MTT assay (a) CHO given treatment with EA fraction
(b) CHO given treatment with AEA fraction (c) HepG2 cells given treatment with EA fraction (d) HepG2 cells given treatment with AEA fraction (e) HeLa cells given treatment with EA
fraction (f) HeLa cells given treatment with AEA fraction (g) MCF-7 given treatment with EA fraction (h) MCF-7given treatment with AEA fraction.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
Accelerys. ADME is an abbreviation in pharmacokinetics and pharma-
cology for absorption, distribution, metabolism, and excretion,which
denes the disposition of a therapeutic compound within a creature.
The four standards all aect the drug levels and kinetics of drug ac-
quaintance to the tissues and hereafter eect the presentation and
pharmacological movement of the compound as a remedy (Malik et al.,
Solubility and partition coecients of compounds were estimated to
show pharmacokinetics. In silico ADMET studies showed results that
were comparable to the results shown by standard antioxidant L-AA.
Results are shown in Tables 35in supplementary les.
3.8. Docking studies
Nowadays, there is strong concern of the anticancer research com-
munity concerning enhancement of the techniques in use. This directed
in particular to a rising extent of drug-enzyme research achieved by
means of valuable techniques superior to contemporary methods used
in the eld. Pharmacokinetics studies screened the phytoconstituents
obtained in the GC-MS analysis as potent drug like candidates.
Compounds have been selected from the relative abundance of their
presence in GC-MS analysis. We performed in silico virtual screening of
all selected compounds to see the receptor ligand interactions using
Auto Dock tools. Best docked interactions are listed in Table 5. Out of
all the compounds capsaicin and piperine when docked with anti-
oxidant enzymes showed the most negative energy of binding. We
identied the active site residues involved in receptor ligand interac-
tions. Structure of compounds and enzymes are shown in Fig. 7.
Fig. 8al represents the interactions.
Capsaicin when docked with SOD (PDBID:4mcm)showed residues
Ser-102,Glu-100 formed hydrogen bonds and residues Lys-75, Lys-128,
Lys-70, Lys-75 provided a strong basic environment. Asp-124,Asp-
76,Glu-78,Glu-100,Val-14,Asn-13,Leu-42,Glu-70,Asn-76 provided a
strong acidic environment. Phe-45 provided with aromatic environ-
ment. Binding energy of the interaction shown was 6.9 kcal/mol.
Piperine when docked with SOD (PDBID:4mcm)showed that Arg-
115 residue was involved in hydrogen bonding and Asp-109,Asp-
83,Glu-77 provided strong acidic environment. Leu-84, Ala-123, Gly-
93, Gly-114, Val-97, Val-5, Leu-126, Leu-144, Gly-93 were involved in
strong hydrophobic interactions. Lys-128, Lys-91, Arg-115, Lys-91pro-
vided strong basic environment Phe-45 provided a strong aromatic
binding. Binding energy shown was 4.7 kcal/mol.
While interacting with CAT (PDBID:1dgb) capsaicin presented Arg-
170, Lys-177, Ser-254, Gln-255, Glu-256 as hydrogen bonded residues.
Glu-255, Asp-259, Asp-124 provided with acidic environment. Residues
Cys-393, Gly-367, Ala-76, Val-116, Lys-169,Gln-168, Ser-120, Ser-122,
Thr-125, Lys-77, Leu-262 provided hydrophobic interacting residues.
Binding energy of interaction was 7.3 kcal/mol.
Fig. 5. Eects of EA and AEA fractions on Curculigo orchioides Gaertn along with standard drug doxorubicin treatment on the cells using DAPI staining (a) HepG2 cells under controlled
conditions. (b) HepG2 cells given treatment with doxorubicin (c) HepG2 cells given treatment with EA fraction (d) HepG2 cells given treatment with AEA fraction (e) HeLa cells under
controlled conditions. (f)HeLa cells given treatment with doxorubicin (g) HeLa cells given treatment with EA fraction (h) HeLa cells given treatment with AEA fraction. fraction (i) MCF-
7 cells under controlled conditions. (j) MCF-7cells given treatment with doxorubicin (k) MCF-7 cells given treatment with EA fraction (l) MCF-7cells given treatment with AEA fraction.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
Similarly piperine interacted with CAT (PDBID:1dgb) with the fol-
lowing amino acid residues. Ala-123 formed hydrogen bond. Asp-257,
Asn-77, Asn-324, Glu-60, Asn-403, Asp-335, Asn-324 provided acidic
environment. Lys-77,Val-73, Ser-120,Vai-126,Ser-68,Arg-68,Arg-
170,His-175, Arg-170, His-166, Asp-348, provided hydrophobic inter-
actions. Phe-326, Tyr-500, Tyr-358, provided aromatic environment.
Binding energy of interaction was 8.2 kcal/mol.
While interacting with GPx (PDBID: 3kij), capsaicin showed polar
interactions with Arg-127, Glu-125, Ileu-136. Residues Glu-161, Asp-
54, Glu-63, Glu-176, Glu-161 provided acidic environment. Residues
Lys-53, Lys-66, Leu-1, Leu-71, Ala-150, Val-60, Ser-61, Leu-71, pro-
vided hydrophobic environment. Phe-106, Phe-156, Phe-50, Tyr-48,
Phe-47, Tyr-65, Phe-106 provided aromatic environment. Binding en-
ergy of the interaction is 5.0 kcal/mol.
Similarly piperine while interacting with GPx (PDBID:3kij) showed
polar interactions with Lys-128, Asn-129, Asp-83, Glu-161, Asp-156,
provided acidic environmer-145,Pro-162,Lys-160,Ser-158,Lys-168, Val-
177 provided hydrophobic interaction. Tyr-130,Phe-162,Phe-153,Phe-
180, Trp-180, Phe-151 provided aromatic interactions. Binding energy
of interaction was 6.2 kcal/mol.
Similar interactions are shown while interacting with GST (PDBID:
4mpg). Capsaicin polar interactions were shown by residues Thr-109,
Arg-239, Arg-242, Asp-104 and hydrophobic interactions were formed
by residues Ala-103, Arg-107, Gly-108, Ileu-243, Pro-244, Ala-241, Lys-
131, and Asn-135. Binding energy of interaction was 7.9kcals/mol.
While piperine interacting with GST (PDBID: 4mpg). hydrogen
bonding was shown by Lys-53 and CSO-105. Arg-117, Arg-242, Ile-243,
Pro-240, CSO-121, GSH-301, Ala-138,Thr-137,Pro-240, Pro-244, Arg-
107, Leu-143, Met-139, His-40 provided hydrophobic interactions
while Trp-145 provided aromatic environment. Binding energy of in-
teraction was 8.2kcals/mol. All the values of binding energies were
comparable to the standard compound L-Ascorbate.
A brief illustration is showed in Fig. 9 showing the mitochondrial
pathways involved during combatting oxidative stress.
4. Conclusion and future perspectives
Over the last decades a substantial number of studies have been
done by the researchers to nd out the correlation between cancer
susceptibility and the expression levels of antioxidant enzymes. As
Fig. 6. (a) Representative western blot of cells showing the expression of Bcl-2, caspase 8 and caspase 3. Con represents controls (untreated cells) for HepG2 HeLa and MCF-7 cells
respectively; β-actin is taken as positive loading control.
Bars represent the densitometric intensity of the indicated protein bands, using image analyzing software. Data are presented as the means ± SD of three independent experiments. For
HepG2 p* < 0.05, ⁄⁄, p** < 0.01,⁄⁄⁄p*** < 0.001,///p**** < 0.0001.; For HeLa p# < 0.05, ⁄⁄, p## < 0.01,⁄⁄⁄p### < 0.001,///p#### < 0.0001, For MCF-7 Pˆ< 0.05, ⁄⁄,
pˆˆ < 0.01,⁄⁄⁄pˆˆˆ < 0.001,///pˆˆˆˆ < 0.0001.
(b) Densitometric intensity of caspase 3 versus the control, where 1st, 4th and 7th bars are respective controls (untreated cells) of HepG2, HeLa and MCF-7 cells; 2nd and 3rd bars are
representatives of HepG2 cell lines treated with EA fraction and AEA fraction; 5th and 6th bars are representatives of HeLa cell lines treated with EA fraction and AEA fraction and 7th and
8th bars are representatives of MCF-7 cell lines treated with EA fraction and AEA fraction respectively.
(c) Densitometric intensity of caspase 8 versus the control, where 1st, 4th and 7th bars are respective controls (untreated cells) of HepG2, HeLa and MCF-7 cells; 2nd and 3rd bars are
representatives of HepG2 cell lines treated with EA fraction and AEA fraction; 5th and 6th bars are representatives of HeLa cell lines treated with EA fraction and AEA fraction and 7th and
8th bars are representatives of MCF-7 cell lines treated with EA fraction and AEA fraction respectively.
(d) Densitometric intensity of Bcl-2 versus the control, where 1st4th and 7th bars are respective controls (untreated cells) of HepG2, HeLa and MCF-7 cells; 2nd and 3rd bars are
representatives of HepG2 cell lines treated with EA fraction and AEA fraction; 5th and 6th bars are representatives of HeLa cell lines treated with EA fraction and AEA fraction and 7th and
8th bars are representatives of MCF-7 cell lines treated with EA fraction and AEA fraction respectively.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
cancer is one of the most intricate ailments and its pathogenic me-
chanism is varied, new studies about expression levels of several anti-
oxidant enzymes will open the new potentials to comprehend the pa-
thogenesis of cancers and therapies. The ndings explained that
fractions and compounds of Curculigo orchioides Gaertn eectively in-
hibited H2O2 mediated oxidative stress by increasing its antioxidant
enzyme content and show cytotoxic potential on cancer cell lines
HepG2, HeLa and MCF-7. Pharmacokinetics studies screened the phy-
toconstituents obtained in the GC-MS analysis as potent drug like
candidates. Further docking studies showed the virtual binding inter-
actions of the phytoconstituents with the antioxidant enzymes chosen
for the study (Prakash Amresh et al., 2013). The binding interactions
consisted of hydrogen bonding, van der Waals forces, aromatic inter-
actions and hydrophobic interactions. From the docking perspective,
direct comparison of inhibitory potency and IC
proles of com-
pounds, revealed that compounds from the plant (capsaicin and
piperine) show binding anity better than the rest of the compounds
with the given antioxidant enzymes. Thus these compounds can prove
to be better antioxidants in relieving oxidative stress by enhancing
antioxidant enzyme defense system that in turn activates the anticancer
enzymes. However further independent studies need to be done on the
antioxidant and anticancerous nature of the individual compounds.
Also, more studies can be focused on the pathways involved at the
cellular levels which can give a brief insight of the active cell machinery
involved for programmed cell death. Also, understanding the molecular
events that control apoptosis in response to anticancer therapies, and
how cancer cells evade apoptotic death, delivers novel openings for a
more balanced approach to grow molecular-targeted treatments for
ghting cancer. Anyhow our studies have suggested that Curculigo
orchioides Gaertn may be useful as a therapeutic agent in preventing
oxidative stress mediated diseases and cancer oncogenesis.
Table 5
Protein ligand interactions.
Protein Complexes Energy (k cal/mol) Interacting Residues
L Ascorbate
Superoxide Dismutase (4mcm) 5.3 ASn-26,Ser-109,Gly108,Cys-117,Asn-65,Val-81,Val-103,Ser-102,Val- 29,Gln22,Phe-20,his110,Ileu112,Ileu-114,Asp101
Catalase (1dgb) 6.6 Phe-326,Asn-324,Asn-397,His-175,Asn-403,Ser120,Asn171,Glu71,Tyr-325,Pro-172,Asp-389,Arg-388,Gly- 118,Ala-
Glutathione Peroxidase (3kij) 5.6 Arg-163,Arg-117,Glu-122,Glu-115,Phe-125,Glu-80,Trp-164,Thr-82,Phe-166,Gln-110,Ala-76,Leu-143,Lys-160,Phe-
Glutathione- s -Transferase
6.5 Arg-239,Gln-12,Gly-106,Pro-13,ileu-112,Leu-114,Val-10,Ser-14,Pro- 73,Arg-107,Thr-109,GSH-301,Leu-7,Trp-
Protein Complexes Capsaicin Interacting Residues
Superoxide Dismutase (4mcm) 6.9 Ser-102,Glu-100,Lys-75,Lys-128,Asp-124,Lys-128,Asp-76,Glu-78,Glu-100,Lys-128,Val-14,Asn-131,Lys-70,Leu-42,Glu-
Catalase (1dgb) 7.3 Arg-170,Lus-177,Ser-254,Gln-255,Glu-256,Glu-255,Asp-259,Cys-393,Gly-367,Ala-76,Val-116,Lys-169,G ln-168,Ser-
Glutathione Peroxidase (3kij) 5 Arg-127,Glu-125,Ileu-136,Phe-106,Ala-150,Pro-135,Leu-71,Ala-150,Val-60,Ser-61,Phe-50,Glu-63,Tyr-48,Phe-47,Tyr-
Glutathione s- Transferase
7.9 Thr-109,Arg-109,Arg-242,Asp-104,Ala-103,Arg-107,Gly-108,Ile-243,Pro-244,Ala-241,Lys-131,Asn-135
Protein Complexes Piperine Interacting Residues
Superoxide Dismutase (4mcm) 4.7 Arg-115,Phe-45,Asp-109,Leu-84,Ala-123,Asp-83,Lys-128,Arg-7,Gly-114,Lys-91,Gly-93,Val-5,Val-97,Glu-77,Leu-
Catalase (1dgb) 8.2 Ala-123,Tyr-500,Lys-77,Val-126,Ser-68,Tyr-358,Asp-257,Ser-120,Val-73,Arg-68,Phe-326,Asn-77,Arg-1 70,His-175,Asn-
Glutathione Peroxidase (3kij) 6.2 Lys-128,Asn-129,Tyr-130,Asp-83,Arg-163,Pro-162,Ser-145,Pro-162,Glu-161,Phe-162,Lys-160,Asp-156,Ser-158,Phe-
Glutathione s- Transferase
8.2 Lys-53,CSO-105,Arg-117,Arg-242,Ile-243,Pro-240,CSO-121,GSH-301,Phe-110,Trp-145,Ala-138,Thr-137,Pro-244,Arg-
Fig. 7. Structure of compounds (a) capsaicin (b) piperine and structure of antioxidant enzymes (a) 4mcm (SOD) (b) 1dgb (CAT) (c) 3kij (GPx) (d) 4mpg (GST).
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
Fig. 8. Docking interactions of capsaicin and piperine with antioxidant enzymes. (a) Interactions of capsaicin with 4mcm (SOD) (b)Interactions of capsaicin with 1dgb (CAT) (c)
Interactions of capsaicin with 3kij (GPx) (d) Interactions of capsaicin with 4mpg (GST) (e) Interactions of piperine with 4mcm (SOD) (f)Interactions of piperine with 1dgb (CAT) (g)
Interactions of piperine with 3kij (GPx) (h)Interactions of piperine with 4mpg (GST).
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
Fig. 8. (continued)
Fig. 9. Illustrated pathway of free radicle quenching by antioxidant defence of cell showing antioxidant nature of the plant Curculigo orchioides Gaertn.
I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
5. Statistical analysis
The Graph Pad Prism computer software, version 6.1 was used for
data analysis. Quantitative data were presented as mean ± SD for
normally distributed data. Means, Student's t-test and ANOVA were
used followed by Post Hoc test for comparison of the groups.
Correlation between quantitative variables was done using Pearson's
correlation coecient (r). All tests were two tailed and considered
signicant at P < 0.05.
Conicts of interest
Authors show no conict of interest.
Ethical approval
All procedures performed in studies involving animals were in ac-
cordance with the ethical standards of the institution or practice at
which the studies were conducted.
Iram Iqbal Hejazi First author of the manuscript and has executed
the major part of the study as collection of the specimen, extraction of
the solvent extracts, phytochemical screening, in vitro antioxidant as-
says, ex vivo antioxidant assays, molecular docking studies, pharmaco-
Rashmin Khanam Has helped in GC-MS analysis of the extracts.
Syed Hassan Mehdi Has contributed in cellular based studies.
Abdul Roouf Bhat Has designed the idea of the study.
M. Moshahid Alam Rizvi Has contributed in cellular studies.
Sonu Chand Thakur Has designed the idea of the study.
Fareeda Athar All work has been done under her supervision.
Iram Iqbal Hejazi acknowledges University Grants Commission
Maulana Azad National Fellowship for the nancial support of the
work. The authors are thankful to UGC scheme-UGC (F. No. 43-172/
2014 (SR))for scal support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
Transparency document
Transparency document related to this article can be found online at
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I.I. Hejazi et al. Food and Chemical Toxicology 115 (2018) 244–259
... Rimpang tumbuhan ini secara tradisional digunakan sebagai antiosteoporosis, antioksidan, estrogenik, neuroprotektif, antibakteri, antiasma, antibronkhitis, antiapoptosis, antikanker, dan juga sebagai diuretik dan afrodisiak (Hejazi et al. 2018). Kandungan kimianya berupa glukosida orcinol memiliki aktivitas antioksidan (Wu et al. 2005), glikosida sikloartan digunakan sebagai obat leukemia (Yokosuka et al. 2010), curculigoside F dapat menghambat virus hepatitis B (HBV) e antigen (HBeAg) pada pengujian dengan galur sel HepG2.2.15 (Zuo et al. 2010) , golongan glikosida klorofenol seperti curculigine M, curculigine N, dan curculigine O mendorong proliferasi osteoblas . ...
... Metabolit sekunder yang banyak dimanfaatkan sebagai antioksidan dan antidiabetes yang dipicu oleh radikal bebas adalah golongan polifenol (Ghasemzadeh dan Ghasemzadeh 2011;Hejazi et al. 2018). Derivat metabolit sekunder sangat bervariasi, sehingga perlu adanya suatu metode untuk menentukan gugus fungsi yang berperan sebagai antioksidan pada spesies radikal bebas. ...
... Metabolit sekunder tumbuhan terutama golongan fenol dan flavonoid banyak dieksplorasi sebagai antioksidan alami, baik secara in vitro maupun in vivo, serta in silico (Hejazi et al. 2018). Senyawa ini, memiliki kemampuan transfer atom hidrogen (HAT) dan transfer elektron (ET) (Apak et al. 2007). ...
Diabetes mellitus (DM) is a disease caused by lacking of insulin production or by the inability of cells to respond to insulin (insulin resistance). According to the International Diabetes Federation, diabetes cases in the world reach 425 millions and are predicted to increase to 625 millions by 2045. The trend of increasing cases and death rates due to diabetes needs a special attention, especially in the pattern of it’s treatment. Diabetes treatment using natural ingredients is one of the most researched fields in the world because it is effective and safe. Curculigo latifolia Dryand. ex W.T. Aiton and Curculigo orchioides Gaertn. belonging to the family Hypoxidaceae, annual herbs with lanceolate-shaped leaves or parallel lanceolate arranged in a rosette, with yellow flowers, very short stems, and have a long cylindrical rhizome. A total of 39 species of this genus are accepted in the World Checklist of Selected Plant Families (WCSP 2020), including these two species. Both species are known as traditional medicinal plants in various tropical regions. Rhizome of Curculigo spp. is one of the raw material sources for traditional medicine to treat DM; this pharmacological effect comes from secondary metabolites. Those compounds are distributed and accumulated in certain secretory structures within the plant. However, the activities of the active compounds in such diverse plant organs are very difficult to be determined in a short time, as well as its pharmacokinetic and pharmacodynamics parameters. In addition, compounds produced under normal conditions in the nature are very low. Therefore, this study aimed to determine the distribution of secretory structures and the producing and/or accumulating sites of the bioactive compounds through histochemical tests, to determine which bioactive compounds contribute the most to diabetes mellitus, especially in antioxidant activity and α-glucosidase inhibition, and to determine their pharmacokinetics and pharmacodynamics parameters. In addition, this research was also carried out to produce callus and micropropagate the plants, as well as to ensure the existence of those bioactive compounds in in vitro cultured callus. Determination of secretory structure using cross sections of fresh samples according to plant anatomical procedures and histochemical analysis using several reagents were performed to detect groups of metabolites. Determination of bioactive compounds was done using an analysis combination on biological activities (antioxidants and α-glucosidase inhibition) with metabolite fingerprint using FTIR and metabolite profiling with UHPLC-Q-Orbitrap HRMS-based metabolomic and chemometric techniques using partial least squares regression analysis (PLSR). Pharmacokinetics and pharmacodynamics parameters were determined using Lipinski's rule of five, pharmacological networks using Cytoscape, and molecular docking with PyRx, PyMOL, and BIOVIA Discovery Studio. Callus production and micropropagation began with explant sterilization using environmental-friendly sterilants. Callus initiation and organogenesis were induced by various concentrations of auxins and cytokinin. Metabolomic analysis based on metabolite profiling using UHPLC-Q-Orbitrap HRMS and chemometric techniques using principal component analysis (PCA) were carried out to identify the compounds in the callus and plantlet’s leaves. The anatomical and histochemical analysis of fresh tissues showed that all organs contained secretory structures that accumulated various metabolites. The secretory structures identified in the roots, rhizomes, petiole, and leaves of these two species were secretory cavities and idioblasts. The group of compounds identified were phenols, alkaloids, terpenes, essential oils, and lipophilic. They were also spread over some common tissues of the organs. Based on metabolomic and chemometric analysis the main compounds contributing in antioxidant and α-glucosidase inhibition activities were notified from the phenol group, such as curculigoside B, orchioside B; 2,4-Dichloro-5-methoxy-3-methylphenol, orcinol glucoside; 1,1-Bis-(3,4-dihydroxyphenyl)-1-(2-furan)-methane; from the terpene group, such as: curculigosaponin G, H, and I; from the norlignan group, (1S,2R)-O-Methylnyacoside; and from the aldehyde group, 5-hydroxymethylfural, while the functional groups included O–H, C=O, C–O, C–H. These compounds were accumulated more abundantly in the leaves of C. latifolia (DLSP) from Sinjai-Palangka and C. orchioides (DOGM) from Gowa-Malakaji. Pharmacokinetic parameters showed that 33 out of the 79 compounds were able to be absorbed properly, while some compounds did not meet the requirements. The latter compounds must be converted into aglycones if they will be used as medicinal substances. The cynanuriculoside ligand A_qt based on pharmacological network analysis and molecular docking was able to interact pharmacodynamically with hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) target via 6NJ7 receptor, resulting an affinity of –12.0 (kcal mol–1), with amino acid residues in the form of Ala 226, Leu 126, Val 180, Tyr 183, Leu 215, Ser 170, Ile 121, and Val 168. The sterilization of explants with the lowest concentrations of sterilizing agents and a short contact time with the explants produced 90% sterile cultures. The best combination of plant growth regulators (PGRs) for callus induction in C. latifolia and C. orchioides were BAP : IBA at 3 : 5 and 5 : 3 mg L–1, respectively. The callus were green and white, with a compact consistency. Those combinations of PGRs also regenerated shoots and roots in both species. The secretory structures found in the callus were secretory cavities and idioblast cells. In the callus of C. latifolia, phenol was identified in the organogenic parts and epithelium cells of the secretory cavities, and the essential oils were in idioblast cells; while C. orchioides’ callus contained phenol in the organogenic parts only. The compounds that had contribution in antioxidant and α-glucosidase inhibition activities, such as 1,1-Bis-(3,4-dihydroxyphenyl)-1-(2-furan)-methane, (1S,2R)-O-Methylnyacoside; 2,4-Dichloro-5-methoxy-3-methylphenol, curculigoside B, curculigosaponin G, H, and I; orchioside B, and orcinol glucoside were also identified in the callus and plantlet’s leaves. Most of them belong to the phenol group. The general conclusion of this study is that histochemical techniques revealed that there were differences in the accumulation sites of compounds among organs of Curculigo spp. Histochemically, phenolic compounds were identified in the rhizome, petiole, and leaves of C. latifolia, while in C. orchioides they were only identified in the rhizome. Phenolics were also found in the organogenic callus of these two species. From the metabolomic-chemometric analysis, compounds that contributed greatly to the antioxidant and α-glucosidase inhibition activities were accumulated in the leaves of both species. From the pharmacological network and molecular docking approaches, cynanuriculoside A_qt, curculigosaponin L_qt, and curculigenin B were confirmed to have potential for the treatment of diabetes mellitus. The compounds found in the plant’s organs of C. latifolia and C. orchioides that contribute greatly in antioxidant and α-glucosidase inhibition activities were also identified in the callus and plantlet’s leaves resulted from in vitro cultures. Some of which even demonstrated higher concentration (peak area) than those of the original plant organs.
... The present study showed that the CO contains a potent scavenger of DPPH free radical and has an EC50 value of 25.6±1.6 µg/mL (Table 03). Previous studies also showed a moderate DPPH radical scavenging capacity of C. curculigo 23 . The methanol extract of C. orchioides rhizomes was found to be moderately effective in scavenging DPPH radicals 30 . ...
... The methanol extract of C. orchioides rhizomes was found to be moderately effective in scavenging DPPH radicals 30 . The study of Hejazi et al., 2018 revealed that the DPPH radical scavenging capacity of aqueous extract of C. orchioides was EC 50 104.8±0.6 23 . Literature indicates that variation of plant secondary metabolites occur due to geographic location and connected environmental factors like temperature, rainfall, soil type, and composition 31 . ...
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Curculigo orchioides Gaertn is a medicinal herb belonging to Amaryllidaceae family and is recognized as a potent source of biologically active compounds with many biological properties. The rhizome of the plant was used to prepare aqueous extracts in order to screen antioxidant and anti-inflammatory capacity. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) were evaluated to determine the phytochemical composition of the extracts. Antioxidant activity was screened using different assays, DPPH, total antioxidant activity, inhibition of protein oxidation, lipid peroxidation and deoxyribose oxidation and ferric ion reducing power. Anti-inflammatory activity was screened using human red blood cell membrane stability test and inhibition of protein denaturation assays. The TPC and TFC were 101.2±1.3 mg GA/g (Gallic Acid /g) and 178.4±2.4 mg EGCG/g (epigallocatechin gallate/g) respectively. The values of EC50 for DPPH, inhibition of deoxyribose oxidation, inhibition of lipid peroxidation, inhibition of protein oxidation and inhibition of BSA denaturation assays were 25.6±1.6, 10.1±1.0, 29.0±0.7, 82.6±3.9 and 32.9±3.5 µg/mL respectively. Further, RCO extract showed less ferric ion reducing ability. CO demonstrated comparable values for EC50 with Diclofenac sodium standard on membrane stabilizing activity (49.7±1.4 and 49.7±1.4 µg/mL respectively). Result of the study suggests that CO exerts antioxidant as well as anti-inflammatory activities by stabilizing biological membranes.
... Các hợp chất polysaccharid từ sâm cau thể hiện tác dụng chống ung thư rõ rệt trên khối u ác tính cổ tử cung ở chuột thử nghiệm, tăng cường đáng kể chức năng miễn dịch, tác động quá trình apoptosis biểu hiện bằng sự gia tăng chỉ số tuyến ức và lá lách. Hơn nữa, các polysaccharid điều chỉnh đáng kể sự biểu hiện của caspase-3, caspase-9 và protein (p53) đối với tế bào HeLa trong ống nghiệm [16,17,18]. ...
... Bằng phương pháp thử nghiệm hoạt tính gây độc và ức chế sự tăng sinh tế bào MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] trên dòng tế bào ung thư gan Hep-G2 (Hepatocellular carcinoma) kết quả Bảng 3.2 cho thấy: Mẫu SC, SC.E biểu hiện hoạt tính mạnh nhất đối với dòng tế bào ung thư Hep -G2 với giá trị IC 50 lần lượt là 37,2 μg/ml và 18,9 μg/ml các mẫu còn lại hoạt tính yếu hoặc không có hoạt tính kết quả này cũng tương đồng với những nghiên cứu đã được công bố [17][18][19]. chất có hoạt tính gây độc hoặc ức chế sự tăng sinh tế bào. Nguyên tắc của phương pháp là gián tiếp xác định hoạt tính của chất thử qua khả năng ức chế enzyme oxidoreductase phụ thuộc NAD(P)H của tế bào. ...
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Cây sâm cau (Curculigo orchioides Gaertn) thuộc họ Hạ trâm (Hypoxidaceae) Curculigoosystemoides Gaertn. (Hypoxidaceae) là cây thân thảo sống lâu năm, thân rễ hình trụ dài. Thân rễ của loại cây này có nhiều dược tính cũng như các tính chất khác như: giải nhiệt, lợi tiểu, kích thích tình dục, bổ trĩ, trị trĩ, tiêu viêm, lở ngứa, chữa bệnh ngoài da, hen suyễn, viêm phế quản, vàng da, ung thư, làm lành vết thương do tiêu chảy …Thử hoạt tính chống oxi hóa bằng các phương pháp loại bỏ các gốc tự do tạo bởi 1,1-diphenyl-2-picrylhydrazyl (DPPH), phương pháp gây độc và ức chế sự tăng sinh tế bào MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] kết quả cho thấy: Cao etyl axetat (SC.E), cao tổng (SC), cao ethanol - Nước (SCM) biểu hiện hoạt tính chống oxi hóa với giá trị SC50 lần lượt là 38,05 µg/ml, 48,03 µg/ml, 94,04 µg/ml. Mẫu SC, SC.E biểu hiện hoạt đối với dòng tế bào ung thư Hep - G2 với giá trị IC50 lần lượt là 37,2 μg/ml và 18,9 μg/ml. Các mẫu còn lại hoạt tính yếu hoặc không có hoạt tính.
... The C. orchioides ethyl acetate and methanolic fraction have exhibited important antioxidant activities by scavenging free radicals. 46,47 The activity was studied in carbon tetrachloride (CCl 4 )-induced hepatopathy in rats, and it was found that the methanolic extract decreased the activity of antioxidant enzymes. 48 The 1,1-diphenyl-2-picrylhydrazyl and ferric reducing antioxidant power assay of the in vitro and in vivo plant extracts have suggested that both leaf and root extracts have potential antioxidant activity. ...
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Curculigo orchioides , commonly called “Kali Musli,” is an endangered medicinal plant commonly found in Asian countries such as India, Japan, China, and Nepal. The plant holds a significant position in Ayurvedic and the Chinese traditional medicine system; it is documented as an aphrodisiac herb. The plant is also reported to be used in the treatment for asthma and jaundice. The botany, traditional uses, phytochemistry, and pharmacological activities to evaluate the plant's importance and relevant information are reviewed and summarized. We discern that a total of 61 phytochemicals are identified and reported in C. orchioides . These belong to the various phytochemical group of glycosides, lignans, polysaccharides, alkaloids, saponins, triterpenes, and aliphatic compounds. The most explored bioactive compound is a phenolic glycoside, curculigoside, isolated from the plant's rhizome. In vitro and in vivo research is conducted globally to provide primary and robust evidence to support this herbal medicine's traditional uses. A large lacuna regarding the mechanisms involved in the biological activity of the plant is evident. There is a need to conduct in-depth studies to understand the relationship between traditional and modern pharmacological uses of C. orchioides.
... species. Its rhizome extracts are used against antioxidative and anti-proliferative (Hejazi et al., 2018) and neuroprotective activity (Ramchandani et al., 2014). Therefore, in the present study, we have carried out the isolation of bioactive compounds from Indian C. orchioides Gaertn. ...
Alzheimer’s (AD) is one of the most common age-related neurodegenerative diseases in the world. Currently it has affected about 33.9 million people and is estimated to triple over the next 5 years. The accumulation of amyloid-beta plaques in the neurons is one of the important characteristic features of AD which leads to the gradual decline of a person’s memory, learning and reasoning ability leading to difficulty in carrying out daily activities. Medicinal plants are the main ingredients of native medicines and have a major role in traditional health care. Of late, medicinal plants are being accepted widely because of fewer side effects compared to synthetic drugs. Moreover, they also meet the requirement of medicine necessary for increasing human pop- ulation. Medicinal plants play a crucial role in the development of new herbal drugs. Curculigo orchioides Gaertn., commonly known as Kali Musli or golden grass, is extensively utilised as a nutritive tonic for strength and treatment of asthma, skin diseases, neurodegenerative diseases, diarrhea, etc. To isolate and characterize the MS-1 compound from Curculigo orchioides Gaertn.,rhizome. The isolated compound was analysed by LC-MS, HPLC, FTIR, and 13 C &1 H��NMR and molecular docking studies. The neuroprotective effect of the MS-1compound was evaluated on the Appl-GAL4 model of D.melanogaster. The MS-1compound showed good neuroprotection, antioxidant activity and also increased the climbing activity and the lifespan of Drosophila. These results might explain the effect of MS-1 compound as a good neuroprotective agent. In the future, it can be used in making anti-Alzheimer drugs.
... In Chinese medicine, Curculigo orchioides Gaertn (COG, "Xian Mao"), it was applied topically for the management of knee and spine joints arthritis, leg fatigue and diarrhoea. COG seems to have antitumor effects and antioxidant, and could be employed as an antiosteoporotic herbal agent, according to latest research (Ramchandani et al., 2014;Vickers, 2017;Hejazi et al., 2018;Cui et al., 2019). COG has been studied in vivo for its antiosteoporotic activities in TCM for the management of postmenopausal women with osteoporosis. ...
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According to World Health Organization (WHO), osteoporosis is a systematic bone disability marked by reduced bone mass and microarchitectural degeneration of osseous cells, which leads to increased bones feebleness and fractures vulnerability. It is a polygenetic, physiological bone deformity that frequently leads to osteoporotic fractures and raises the risk of fractures in minimal trauma. Additionally, the molecular changes that cause osteoporosis are linked to decreased fracture repair and delayed bone regeneration. Bones have the ability to regenerate as part of the healing mechanism after an accident or trauma, including musculoskeletal growth and ongoing remodeling throughout adulthood. The principal treatment approaches for bone loss illnesses, such as osteoporosis, are hormone replacement therapy (HRT) and bisphosphonates. In this review, we searched literature regarding the Traditional Chinese medicines (TCM) in osteoporosis and bone regeneration. The literature results are summarized in this review for osteoporosis and bone regeneration. Traditional Chinese medicines (TCM) have grown in popularity as a result of its success in curing ailments while causing minimal adverse effects. Natural Chinese medicine has already been utilized to cure various types of orthopedic illnesses, notably osteoporosis, bone fractures and rheumatism with great success. TCM is a discipline of conventional remedy that encompasses herbal medication, massage (tui na), acupuncture, food, and exercise (qigong) therapy. It is based on more than 2,500 years of Chinese healthcare profession. This article serves as a comprehensive review summarizing the osteoporosis, bone regeneration and the traditional Chinese medicines used since ancient times for the management of osteoporosis and bone regeneration.
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Dexamethasone, a synthetic glucocorticoid drug, is widely used as an anti-inflammatory/anti-allergic agent worldwide. Several previouse studies suggested that in the inflammatory or enriched free radical condition, Dexamethasone directly bind to DNA leading to DNA brekage/remodeling and subsequently cancer or other DNA breakage related disorders. So, for the first time, we computationally and experimentally investigated the interaction of Dexamethasone drug, alone and in combination with H 2 O 2 , ascorbic acid, iron and copper, with a routine supercoiled plasmid DNA. In the next step, the intrinsic DNA binding constant (k d ) was calculated using UV absorption titration method via Dexamethasone interaction with calf thymus DNA (ctDNA). Also, interaction of the drug with antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase 4, glutathione reductase was investigated by molecular docking methods. Interestingly, our in-vitro study demonstraded that dexamethasone binds to DNA by binding energy of -5.35 kcal/mol. Our study also indicatd that dexamethasone/DNA interaction lead to no DNA breakage while, its combination with the mineral supplies cause to DNA damage/breakage. This results are consistent with our Docking study that indicated dexamethasone stungly bind to DNA and the catalytic site of glutathione peroxidase 4, FAD-binding site of the glutathione reductase, the active site of the superoxide dismutase and NADPH binding residues of the catalase enzyme. Therefore, we hypothesis that Dexamethasone may indirectly make DNA damages by inhibiting antioxidant defense enzymes and causing oxidative stress in cells.
Introduction Chronic myeloid leukemia (CML) is a myeloproliferative hematological malignancy wherein the increased relapsed cases and lack of drug effectiveness generates a need to identify an effective treatment regimen. A unani formulation, Habb-e-Asgandh (HEA) is being used for treatment of ailments such as arthritis, however, its role in CML therapeutics has not been reported yet. Hence, anti-leukemic efficacy of HEA was assessed alone or in combination with standard chemotherapeutic, imatinib in CML cell line K562. Methods The anti-leukemic effect of HEA was evaluated on cell cytotoxicity by MTT assay, cell cycle analysis by propidium iodide staining, apoptotic assay by Annexin-V assay, mitochondrial membrane potential (MMP) by JC1 staining. Further, effect of drug treatment on oxidative stress and cell migration was determined by ORAC assay and transwell migration assay respectively. Lastly, changes in angiogenic marker (VEGF) and inflammatory marker (IL-6) were examined by ELISA. Results HEA induced cytotoxicity of leukemic cells which increased further when co-treated with imatinib. The combinatorial treatment of HEA with imatinib potentiates anti-tumor effect by inducing cell apoptosis with disruption of MMP even at low concentrations. Subsequent studies revealed that tumor killing property of HEA increased the ROS generation. Furthermore, HEA in combination with imatinib dramatically reduced cell migration along with expression of VEGF and IL-6. Conclusion This maiden study demonstrated that HEA possesses anti-leukemic potential in vitro. This formulation potentiates the effect of imatinib even at low concentrations suggesting the utilization of HEA as a novel adjuvant therapy for the better management of CML in future.
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Correct species identification is crucial for ensuring the quality, safety, and efficacy of herbal medicine. Market research indicates that Curculigo glabrescens Rhizoma (CGR) was the major counterfeit of the medicine Curculigo orchioides Rhizoma (COR). To accurately discriminate COR and CGR remains a challenge, and it becomes even more difficult when the herbs have been heavily processed into a powder. In this work, combined with high performance liquid chromatography analysis, a novel component in CGR was discovered, and two stable isotopes (N%, C%, δ15N, δ13C) and nineteen mineral elements were determined along with multivariate statistical analysis to distinguish the authentic COR samples and counterfeit CGR samples. The results showed that there were significant differences between the mean value of N%, δ15N and δ13C according to the botanical origins. In addition, these two species can be differentiated by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) analysis. A linear discriminant analysis (LDA) model with a good classification rate (100%) and cross-validation rate (100%) was established. Hence, stable isotope and mineral element contents combined with chemometrics analysis could be considered as an effective and reliable method for discriminating the source species of COR and CGR.
Ethnopharmacological relevance The plants of genus Curculigo are divided into the Section Curculigo and the Section Capitulata, which are mainly distributed in southeastern and southwestern China. Various ancient chinese books record that these plants were used as an important herb for tonifying kidney yang. Traditional Chinese medicine often draws on this property to treat depression syndrome. Thus genus Curculigo has potential for the treatment of neurodegenerative diseases (ND). The study showed that phenolics were the main characteristic components of plants in the Section Curculigo, represented by orcinol glucoside and curculigoside; the norlignans, with Ph-C5-Ph as the basic backbone, were the main characteristic components of the Section Capitulata. However, there is a lack of sufficient scientific evidence as to whether these two types of ingredients have neuroprotective effects. Aim of the study To determine the neuroprotective effects of phenolics and norlignans in genus Curculigo on human neuroblastoma cells SH-SY5Y. To discuss their structure-activity relationship and screen for compounds with high activity and neuroprotective effects. To reveal that the amelioration of endoplasmic reticulum (ER) stress by two classes of compounds is mediated by the PERK/eIF2α/ATF4 pathway. Materials and methods The cytotoxicity of 17 compounds was assayed by MTT. SH-SY5Y cells were damaged by corticosterone (Cort) (200 μM) for 24 h and then co-administered with 17 compounds (0.1–100 μM) and Cort (200 μM) for 24 h. Cell survival was determined by MTT assay. Apoptosis rate, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels were detected using flow cytometry. Intracellular Ca²⁺ levels were detected using a fluorescent probe. Cellular mitochondrial and ER damage was observed using transmission electron microscopy (TEM). ER stress and apoptotic pathway-related proteins (BiP, CHOP, cleaved caspase-3, cleaved caspase-9, Bax/Bcl-2), and the expression level of PERK/eIF2α/ATF4 pathway was measured via western blot (WB). Results The experimental data showed that Cort treatment of SH-SY5Y cells resulted in decreased cell survival and increased apoptosis, mitochondrial depolarization, ROS, and intracellular Ca²⁺ levels. The co-action of 17 compounds and Cort for a period of time significantly increased cell survival. Compounds 3, 7, 12, 13 also reduced apoptosis rate, mitochondrial depolarization, ROS and intracellular Ca²⁺ levels in the subsequent experiments. In addition, TEM observed that Cort caused mitochondrial and ER damage, and the damage was improved after treatment. WB analysis obtained that Cort increased the expression of apoptotic and ER stress-related proteins and activated pathway expression. However, in the presence of compounds 3, 7, 12, 13, the expression of BiP, CHOP, cleaved caspase-3, cleaved caspase-9, and Bax/Bcl-2 was significantly reduced, and the phosphorylation of PERK and eIF2α and the expression of ATF4 were inhibited. Conclusion This study found that one phenolic (3) and three norlignans (7, 12, 13) from genus Curculigo have significant neuroprotective effects. The results of the structure-activity relationship indicated that the glucosyl polymeric norlignans and the phenolics with benzoic acid as the parent nucleus were more active. The neuroprotective effect of three norlignans is the latest discovery. This finding has important research value in the field of prevention and treatment of neurodegenerative diseases.
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Piperazine scaffolds or 2-Azetidinone pharmacophores have been reported to show anti-cancer activities and apoptosis induction in different types of cancer cells. However, the mechanistic studies involve in induction of apoptosis addressing these two moieties for human cervical cancer cells remain uncertain. The present study emphasizes on the anti-proliferating properties and mechanism involved in induction of apoptosis for these structurally related azoles derivatives in HeLa cancer cells. 1-phenylpiperazine clubbed with 2-azetidione derivatives (5a-5h) were synthesized, characterized using various spectroscopic techniques and evaluated for their in -vitro anti-proliferative activities and induction of apoptosis. Further, we also evaluated oxidative stress generated by these synthetic derivatives (5a-5h). Cell viability studies revealed that among all, the compound N-(3-chloro-2- (3-nitrophenyl)-4-oxoazetidin-1-yl)-2-(4-phenylpiperazin-1-yl) acetamide 5e remarkably inhibited the growth of HeLa cells in a concentration dependent manner having IC50 value of 29.44±1.46 µg/ml. Morphological changes, colonies suppression and inhibition of migration clearly showed the antineoplasicity in HeLa cells treated with 5e. Simultaneously, phosphatidylserine externalization, DNA fragmentation and cell-cycle arrest showed ongoing apoptosis in the HeLa cancer cells induced by compound 5e in concentration dependent manner. Additionally, generation of intracellular ROS along with the decrease in mitochondrial membrane potential supported that compound 5e caused oxidative stress resulting in apoptosis through mitochondria mediated pathway. Elevation in the level of cytochrome c and upregulation in expression of caspase-3 clearly indicated the involvement of the intrinsic pathway of programmed cell death. In brief; compound 5e could serve as a promising lead for the development of an effective antitumor agent.
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Cancer is a daunting global problem confronting the world's population. The most frequent therapeutic approaches include surgery, chemotherapy, radiotherapy, and more recently immunotherapy. In the case of chemotherapy, patients ultimately develop resistance to both single and multiple chemotherapeutic agents, which can culminate in metastatic disease which is a major cause of patient death from solid tumors. Chemoresistance, a primary cause of treatment failure, is attributed to multiple factors including decreased drug accumulation, reduced drug-target interactions, increased populations of cancer stem cells, enhanced autophagy activity, and reduced apoptosis in cancer cells. Reprogramming tumor cells to undergo drug-induced apoptosis provides a promising and powerful strategy for treating resistant and recurrent neoplastic diseases. This can be achieved by downregulating dysregulated antiapoptotic factors or activation of proapoptotic factors in tumor cells. A major target of dysregulation in cancer cells that can occur during chemoresistance involves altered expression of Bcl-2 family members. Bcl-2 antiapoptotic molecules (Bcl-2, Bcl-xL, and Mcl-1) are frequently upregulated in acquired chemoresistant cancer cells, which block drug-induced apoptosis. We presently overview the potential role of Bcl-2 antiapoptotic proteins in the development of cancer chemoresistance and overview the clinical approaches that use Bcl-2 inhibitors to restore cell death in chemoresistant and recurrent tumors.
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Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
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Background: Cancer has become one of the global health issues and it is the life-threatening disease characterized by unrestrained growth of cells. Despite various advances being adopted by chemotherapeutic management, the use of the current anticancer drugs such as Doxorubicin, Asparginase, Methotrexate, Vincristine remains limited due to high toxicity, side effects and developing drug resistance. Apoptosis is a crucial cellular process and improper regulation of apoptotic signaling pathways may lead to cancer formation. Subsequently, the synthesis of effective chemotherapeutic agents that can induce apoptosis in tumor cell has emerged as a significant approach in cancer drug discovery. Methods: The goal of this work is to develop a potential antitumor agent exerting significant inhibitory effects on cancer cell and low cytotoxicity, for which we focused on the structural features of 1,3,4-oxadiazoles as it a privileged scaffold in modern medicinal chemistry and have the ability to inhibit growth factors, enzymes and kinases potentially involved in the attainment of cellular immortality and carcinogenesis. Result: In vitro MTT screening assay showed the compound 5-aminophenyl-2-butylthio-1,3,4-oxadiazole (5e) showing the highest inhibitory effect against MCF-7 cancer cell with IC50 value 10.05 ± 1.08 µM while it is much safer and less toxic on normal cell line (HEK-293). The dose-dependent treatment of MCF-7 cells with 5e resulted in inhibition of cell migration in the wound healing assay. The flow-cytometry analysis showed the cells arrested in G0/G1 phase of the cell cycle. Compound 5e induced apoptosis of MCF-7 cells was characterized using DAPI staining and Annexin V-PE/7-AAD dual binding assay. Reduction of NBT by compound 5e showed a reduced generation of ROS. Western blotting studies showed high activation of apoptotic protein Caspase3 and decrease in expression of anti-apoptotic protein BCL-2. Conclusion: Based on the results of in vitro studies, it could be concluded that compound 5e showed a significant inhibitory growth effect on MCF-7 cells and have the potential to be developed as lead molecule and further structural modifications may result in promising new anticancer agents.
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Plant-derived substances (phytochemicals) are well recognized as sources of pharmacologically potent drugs in the treatment of several oxidative stress related disorders. Our study aims to evaluate the antioxidant and apoptotic effects of Glycyrrhiza glabra L. in both cell free and cell culture system. Plant fractions have been prepared with hexane, chloroform, ethyl acetate, methanol and water and their antioxidant properties are reviewed. Potent antioxidant activity has been well established in both in vitro and in silico studies which is believed to be responsible for the anticancerous nature of the plant. Results obtained indicate that methanol fraction of G. glabra L. exhibited maximum scavenging activity against DPPH and nitric oxide free radicals comparable to standard antioxidant L-AA. Administration of methanol fraction also considerably reduced the malondialdehyde produced due to lipid peroxidation in mammalian liver tissues. Moreover, the levels of antioxidant enzymes SOD, CAT, GST, GPx and GR in the oxidative stress induced tissues were refurbished significantly after treatment with plant’s methanol fraction. Moreover, methanol fraction was found to be nontoxic to normal human cell line whereas it inhibited cancer cells HeLa and HepG2 considerably. Apoptosis was established by DAPI fluorescent staining and western blot analysis of pro apoptotic protein caspase-8, caspase-3 and anti-apoptotic protein Bcl-2.There is an up regulation in the levels of pro apoptotic caspase-8 and caspase-3 and down regulation of anti-apoptotic Bcl-2. Furthermore, GC–MS analysis of the methanol fraction revealed the presence of many compounds. In silico experiments using Autodock 4.2 tools showed strong affinity of plant compounds towards antioxidant enzymes (proteins) thus validating with the conclusions of antioxidant enzyme assays and establishing a role in cancer pathogenesis.
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Plant-derived substances (phytochemicals) are well recognized as sources of pharmacologically potent drugs in the treatment of several oxidative stress related disorders. Our study aims to evaluate the antioxidant and apoptotic effects of Glycyrrhiza glabra L. in both cell free and cell culture system. Plant fractions have been prepared with hexane, chloroform, ethyl acetate, methanol and water and their antioxidant properties are reviewed. Potent antioxidant activity has been well established in both in vitro and in silico studies which is believed to be responsible for the anticancerous nature of the plant. Results obtained indicate that methanol fraction of G. glabra L. exhibited maximum scavenging activity against DPPH and nitric oxide free radicals comparable to standard antioxidant L-AA. Administration of methanol fraction also considerably reduced the malondialdehyde produced due to lipid peroxidation in mammalian liver tissues. Moreover, the levels of antioxidant enzymes SOD, CAT, GST, GPx and GR in the oxidative stress induced tissues were refurbished significantly after treatment with plant’s methanol fraction. Moreover, methanol fraction was found to be nontoxic to normal human cell line whereas it inhibited cancer cells HeLa and HepG2 considerably. Apoptosis was established by DAPI fluorescent staining and western blot analysis of pro apoptotic protein caspase-8, caspase-3 and anti-apoptotic protein Bcl-2.There is an up regulation in the levels of pro apoptotic caspase-8 and caspase-3 and down regulation of anti-apoptotic Bcl-2. Furthermore, GC–MS analysis of the methanol fraction revealed the presence of many compounds. In silico experiments using Autodock 4.2 tools showed strong affinity of plant compounds towards antioxidant enzymes (proteins) thus validating with the conclusions of antioxidant enzyme assays and establishing a role in cancer pathogenesis.
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Cardiovascular disease is a compound name for clusters of disorders afflicting the heart and blood vessels; it is assuming an increasing role as a major cause of morbidity and mortality. Unhealthy practices such as smoking, high intake of saturated fat and cholesterol, diabetes and physical inactivity are predisposing factors. The risk factors cause alteration in vascular integrity, compromised membrane integrity, increase free radical generation and reduced endogenous antioxidant system resulting in oxidative stress. Substance with ability to maintain vascular integrity, prevent, or reduce radical formation are able to treat cardiovascular disease. Conventional drugs in use to this effect are with side effect and as alternative, medicinal plants are increasingly gaining acceptance from the public and medical professionals. Reports have shown that bioactive compounds in plants with antioxidant, anti-inflammatory, ability to protect vascular endothelium, prevent lipid oxidation, and augment endogenous antioxidant system are cardioprotective. Phenolics and flavonoids in medicinal plants have been widely reported to play these major roles. This study reviewed the role of bioactive compounds in medicinal plants using a wide range database search.
Lianzhi Cui,1,2 Yawei Zhao,1 Yue Pan,1 Xiao Zheng,1 Dan Shao,1 Yong Jia,3 Kan He,1 Kun Li,3 Li Chen1,3 1Department of Pharmacology, College of Basic Medical Sciences, Jilin University, Changchun, 2Clinical Laboratory, Jilin Cancer Hospital, Changchun, 3School of Nursing, Jilin University, Changchun, China Abstract: Recurrence is one of the major causes of high mortality in ovarian cancer. However, the mechanism of ovarian cancer recurrence after chemotherapy has not been fully understood. In the present study, we investigated the effect of chemotherapy-induced tumor microenvironment on the proliferation of SKOV3 cells. We have shown that SKOV3 cells repopulated faster in the culture medium from apoptotic SKOV3 ovarian cancer cells after 24 h of etoposide phosphate (VP-16) treatment. We found that during apoptosis, cleaved caspase 3 could activate cytosolic calcium-independent phospholipase A2, which stimulated the release of arachidonic acid (AA) and triggered the production of prostaglandin E2 (PGE2). An increased level of phosphorylated focal adhesion kinase (FAK) subsequently facilitated the reproliferation of SKOV3 cells, and VP-16-induced repopulation effects were partially reversed by the FAK inhibitor PF562271. Furthermore, the plasma AA-to-PGE2 ratio and tumoral FAK expression of ovarian cancer patients after chemotherapy were significantly lower than those before chemotherapy. Taken together, our results indicate that chemotherapy-induced apoptotic cancer cells can produce PGE2-enriched microenvironment through caspase 3-mediated AA metabolic pathway, which could lead to the abnormal activation of FAK and eventually accelerate the repopulation of SKOV3 cells. Our study provides novel insight into a mechanism that may be utilized to prevent ovarian cancer recurrence in response to chemotherapy. Keywords: ovarian cancer, repopulation, chemotherapy, apoptosis, FAK
Piperine (PP), an alkaloid from black and long peppers (Piper nigrum Linn &Piper longum Linn), exhibits antitumor activities in vitro and in vivo. We investigated the ability of piperine (PP) to reverse the drug resistance of human cervical cancer cells. In our study, the cervica cancer cells resistant to mitomycin-C (MMC) treatment were used. We found the growth inhibitory effects of piperine on human cervical cancer cell, which were resistant to MMC. Piperine and MMC co-treatment resulted in a dose-dependent suppression of the cell proliferation. Decreasing of phosphorylated-signal transducer and activator of transcription (p-STAT3) was linked to the suppression of p65 by PP and MMC combinational treatment. Additionally, the presence of PP potentiated the effects of MMC on apoptosis induction in cervical cancer cells with drug resistance, which was dependent on Bcl-2 inhibition. The pro-apoptotic proteins of Bax and Bid were up-regulated, accompanied with Caspase cleavage. Moreover, in mice xenograft models, the combined therapy inhibited tumor growth compared to the PP or MMC mono-therapy group. Our data indicated a novel therapeutic strategy of PP to potentiate MMC-induced anti-tumor effect on cervical cancer cells with drug resistance through blocking p-STAT3/p65 and Bcl-2 activation.
Glaucoma is the leading cause of irreversible blindness worldwide, affecting 64.3 million people. An estimated 60.5 million people are affected by primary open angle glaucoma globally, and this will increase to 111.8 million by 2040. The definition of glaucoma has evolved greatly over time. Although multiple risk factors such as ischemia, inflammation, myopia, race, age and low ocular perfusion pressure may play a role, intraocular pressure (IOP) is still the main risk factor we can easily identify and modify. Currently, both medical and surgical interventions aim to reduce IOP. Effective IOP reduction controls and prevents the progression in many cases of glaucoma. Although this multifactorial disease's true pathophysiology is difficult to elucidate, physiologic mediators including nitric oxide (NO) are being evaluated as novel ways to impact progression by both lowering IOP and improving optic nerve head perfusion. Latanoprostene bunod 0.024% is an emerging therapeutic agent that has shown promise in clinical trials. As a nitric oxide-donating prostaglandin F2-alpha receptor agonist, it has proven to effectively, and with good tolerability, reduce IOP in glaucoma and ocular hypertensive patients. Latanoprostene bunod capitalizes on NO's ability to modulate the conventional aqueous humor outflow system, directly improving outflow through the trabecular meshwork, Schlemm's canal and distal scleral vessels. Importantly, targeting the conventional outflow tissues with NO-donating drugs represents an opportunity to restore outflow function, which will most likely have a beneficial consequence of additional IOP-lowering effects with dampening of diurnal and other IOP fluctuations, the benefit of a healthy trabecular meshwork.