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Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In vitro

  • Özel Esencan Hastanesi, İstanbul, Turkey

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Gossypetin is a flavonoid which has anti-mutagenic, anti-atherosclerotic, antioxidant, as well as cytoprotective and antimicrobial effects. The objective of this study was to investigate the cytoprotective role of gossypetin (GP) against cyclophosphamide (CP) toxicity in the human lymphocyte culture. Cytotoxic, necrotic and apoptotic effects of CP (1mM), GP (25, 50 and 100 µM) and combination of them (CP+GP) were studied by using MTT assay and Flow cytometry analysis. It was detected that CP significantly decreased cell viability rate via arresting cell cycle and increasing apoptosis/necroptosis. However, GP treatment reduced negative effects of CP at different concentrations. The most effective concentration of GP against CP toxicity was 25 µM. This concentration GP increased live cell number and cell viability, in addition decreased necrotic and late apoptotic cell quantity which were treated with CP. These results suggest that GP could attenuate the cytotoxic effects of CP and protect the healthy cells when it is used during chemotherapy.
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Medicinal chemistry
Ustunsoy et al., Med chem 2016, 6:2
Research Article Open Access
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 88
*Corresponding author: Seyfettin Ustunsoy, Medical Faculty, Medical
Biochemistry Department, Fatih University, 34500, Istanbul, Turkey, Tel: +90-212-
8663300/7021; E-mail:
Received January 25, 2016; Accepted February 17, 2016; Published February
22, 2016
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin
against Cyclophosphamide Toxicity in Human Lymphocyte Culture In vitro. Med
chem 6: 088-092. doi:10.4172/2161-0444.1000330
Copyright: © 2016 Ustunsoy S, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Protective Role of Gossypetin against Cyclophosphamide Toxicity in
Human Lymphocyte Culture
In vitro
Ustunsoy S1*, Akal ZU2 and Alpsoy L3
1Medical Faculty, Medical Biochemistry Department, Fatih University, 34500, Istanbul, Turkey
2Science and Art Faculty, Biology Department, Fatih University, 34500, Istanbul, Turkey
3Medical Faculty, Medical Biology Department, Fatih University, 34500, Istanbul, Turkey
Gossypetin is a avonoid which has anti-mutagenic, anti-atherosclerotic, antioxidant, as well as cytoprotective
and antimicrobial effects. The objective of this study was to investigate the cytoprotective role of gossypetin (GP)
against cyclophosphamide (CP) toxicity in the human lymphocyte culture. Cytotoxic, necrotic and apoptotic effects
of CP (1mM), GP (25, 50 and 100 µM) and combination of them (CP+GP) were studied by using MTT assay and
Flow cytometry analysis. It was detected that CP signicantly decreased cell viability rate via arresting cell cycle and
increasing apoptosis/necroptosis. However, GP treatment reduced negative effects of CP at different concentrations.
The most effective concentration of GP against CP toxicity was 25 µM. This concentration GP increased live cell
number and cell viability, in addition decreased necrotic and late apoptotic cell quantity which were treated with CP.
These results suggest that GP could attenuate the cytotoxic effects of CP and protect the healthy cells when it is used
during chemotherapy.
Keywords: Cyclophosphamide; Gossypetin; Anticancer; Apoptosis;
Necrosis; Cytotoxicity; Cell cycle
Flavonoids are natural ingredients of plants and their by-products
obtained by dierent methods like heating, boiling, fermenting, etc.
ey have been used as therapeutic solution to the health problems by
humans for centuries even though they do not know the exact acting
mechanism. Nowadays, there are approximately 4000 plant originated
potentially therapeutic natural avonoids in the market. ey are
classied into dierent groups according to their chemical structure
[1]. ese bioactive compounds have crucial roles at regulating
important metabolic signaling pathways during physiological or
pathophysiological cascades in human body [2,3]. Hence, there
are hundreds of studies in the literature which have revealed the
signicance of these nutrients at both providing the healthy life and
preventing/curing diseases such as atherosclerosis, diabetes, metabolic
syndrome, neurodegenerative diseases and cancer by means of anti-
inammatory or antioxidant properties. Moreover, avonoids could
increase or decrease the eect of key regulatory enzymes of metabolism
via changing their expressions at DNA levels [2,3].
Despite this huge number of avonoids, quercetin is the most
studied one due to its unique chemical and physiological properties
in addition to broad prevalence in plants. Furthermore; luteolin,
kaempferol, myricetin, catechin, rutin, setin, thymoquinone and
gossypetin are the other remarkable bioactive compounds [4,5].
Gossypetin is usually extracted from the owers of the hibiscus species.
is molecule is named in chemistry as 3,5,7,8,3',4' hexahydroxy
avone. Previous studies revealed that gossypetin has anti-mutagenic,
anti-atherosclerotic, antioxidant, cytoprotective and antimicrobial
eects likewise quercetin and luteolin because of its unique chemical
structure [6]. Ngai reported that gossypetin has antiproliferative
eects on breast cancer (MCF-7) and hepatocellular cancer (HepG-
2) cell lines. Especially between 25 and 100 µM GP is more eective
on MCF-7 and HepG-2 [7]. In addition, a newly published paper
has shown that gossypetin could protect cells against both radiation
induced oxidative stress and radiation induced cellular damage via
decreasing reactive oxygen species (ROSs) [8]. Previous studies have
shown that ROSs make vital modications on biomolecules like
lipids, proteins and DNAs which change their morphology, biological
functions, life spans and leads to chronic progressive diseases like
atherosclerosis, cancer, inammation, neurodegenerative diseases, and
aging [9]. At this point, avonoids have crucial importance because
of their antioxidant properties. ese bioactive substances can both
decrease the production of reactive oxygen species and neutralize their
toxic eects [9,10]. Moreover, Chen et al. have demonstrated another
important eect of the gossypetin which inhibits LDL cholesterol
oxidation, intracellular lipid accumulation and foam cell formation
through intra cellular signaling molecules that have signicant roles in
the development of atherosclerosis such as PPRα, PPRγ, ABCA1 and
CD36 [11]. In addition, there are recently published papers suggesting
new treatment regimens including antioxidant avonoid molecules
either for protecting the healthy cells against antineoplastic, cytotoxic,
immunosuppressive agents or as a therapeutic agent alone [12].
Cyclophosphamide (CP) is a prodrug that is commonly used in the
treatment of a broad spectrum of cancers and some sort of autoimmune
diseases. Its mechanism of action depends on the active metabolite
of 4-hyroxycyclophosphamide which is produced in the liver by
cytochrome enzyme complexes [13,14]. In summary, this molecule
adds an alkyl group to the DNA and prevents cellular replication.
As a result, CP directs the cells to the apoptosis [13,14]. Although
immunosuppression and cytotoxicity are unwanted side eects of CP,
these properties also make this drug a choice in the treatment of severe
autoimmune diseases for reducing the disease burden and improving
the patient's general health status [15].
In this study, we aimed to investigate the protective eects of GP
against cellular damage which is formed by CP. As far as we know, this
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 89
is the rst paper in the literature that is revealing the protective eect of
the GP during CP chemotherapy.
Materials and Methods
All products used for cell culture studies (RPMI-1640,
phytohaemaglutinin (PHA), fetal bovine serum (FBS), phosphate
buer saline (PBS), Ficoll, L-glutamine and penicillin–streptomycin
(PS) were purchased from Biochrom AG (Mannheim, Germany) and
Biological Industries (Kibbutz Beit Haemek, Israel). Cell proliferation
assay kit (MTT) and Annexin-V CY3 Apoptosis Detection Kit Plus
(containing SYTOX Green Dye) were purchased from Biovision
(Mannheim, Germany), Ficoll-Hypaque, Propidium iodide (Sigma
Aldrich, St Louis, Missouri, USA). Gossypetin is obtained from
ChromaDex (Irvine, USA). All other chemicals used for cell culture
studies and ow cytometry analysis were of reagent grade quality
obtained from commercial sources.
Lymphocyte isolation and culture of PMBC
e peripheral blood mononuclear cells (PBMCs) were isolated
from heparinized blood samples of a healthy volunteer person by
centrifugation at 2500 rpm for 25 min at room temperature according
to the Ficoll-Hypaque density gradient method. All donors gave full
written informed consent. e cloudy layer (containing thrombocyte,
lymphocyte, monocyte, etc.) between the plasma and coll solution
was separated with the help of a pipet. Lymphocytes were washed two
times with PBS, and pellet was suspended in RPMI-1640 media. e
number of viable PMBC cells was determined with the trypan blue
method. Adjusted cell suspension density was equal to 1 × 106 cell/
mL. e PBMCs were cultured on a 96 micro-well plate at a density
of 1 × 106 cell/mL and incubated with CP (1 mM), GP (25 µM, 50 µM
and 100 µM) and CP(1mM)+GP (25, 50 and 100 µM) combinations
supplemented with 2 mM/l L-glutamine, 10% FBS and antibiotics
(penicillin and streptomycin) for 4 days.
MTT assay
e cytotoxicity was measured by MTT assay. A cell viability test
of GP was performed on cultured human lymphocytes to detect the
maximal non-toxic concentration according to the manufacturer’s
protocol. A total of 1 × 104 cells were seeded on each well of a 96-well
plate and cultured for 24 hours. e cells were treated with dierent
concentra tions of CP (1mM), GP (25, 50 and 100 µM) and CP+GP
(25, 50 and 100 µM) combinations in a serum-free medium for 24
and 48 hours. Gossypetin concentrations were selected according to
previous studies [7]. At the end of the incubation process/time, the
medium containing compound was exchanged with a fresh RPMI-
1640 medium, and 10 μL of the reconstitute MTT mixture kit reagent
were added to each well. Aer cultur ing for 4 hours, the absorbance of
the sample was measured by using a microtiter plate reader (Bio-TEK)
at 590 nm. e viability of the cells was determined as the percentage of
viable cells of the untreated control and experiments were performed
in triplicate, and data are expressed as mean of triplicate measurements
(mean ± SD) in percentage of untreated cells (100%).
Measuring apoptosis using ow cytometry
Apoptotic and necrotic cells were identied with Annexin V-Cy3
and Sytox green according to the manufacturer’s protocol, respectively.
PMBC cells (2 × 104 cells/well) were exposed to CP (1 mM), GP (25, 50
and 100 µM) and CP (1 mM) + GP (25, 50 and 100 µM) combinations
for 24 h, aer trypsinizaiton cells were collected and then washed with
PBS, and incubated with 5 µl Annexin V-Cy3 and 1 µl Sytox green for 10
min in the dark. Cells incubated in the CP and GP free growth medium
were used as the negative control. Aer staining a cell population with
Annexin V-Cy3 and Sytox Green dye in the provided binding buer,
cell counting data were acquired BD Accuri C6 soware. Apoptotic
cells show red uorescence (Cy3), dead cells show green uorescence
(sytotox green) and live cells show little or no uorescence. ese
populations can be distinguished easily by ow cytometry detecting in
the FL1 (FITC) channel (Ex. 488 nm/Em. 530 nm) for Sytox Green dye
and FL2 (PE) channel for Annexin V-Cy3 (Ex. 543 nm/Em. 570 nm).
Cell cycle proling with PI staining
Aer trypsinization, cells were collected into 5 ml propylene tubes
(1 × 106) and centrifuged at 15,000 rpm, then stained with propidium
iodide (PI) and analyzed by using BD Calibur ow cytometer, and
data were evaluated with BD Accuri C6 soware. In summary, cells
were washed with 2 ml PBS then centrifuged and supernatant was
discarded. In addition, cells were xed by adding 1 mL of 70% ice
cold EtOH slowly on the pellet while vortexing to avoid cell clumping.
Cells were incubated on ice for 30 minutes then centrifuged to get
rid of the EtOH. Cell pellet was washed with PBS and centrifuged
again. Supernatant was discarded and cell pellet was re-suspended
in 1 mL of PI staining solution. Cells were incubated at 37°C for 40
minutes in PI staining solution and centrifuged again to remove the
staining solution. Supernatant was discarded and the cell pellet was re-
suspended in 500 μl PBS then analyzed at BD Calibur ow cytometer.
All the centrifugations were carried out at 2000 g for 5 minutes.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD).
Statistical dierences between experimental groups were determined
using Student’s t-test. All statistical analyses were performed using
GraphPad Prism 5 and SPSS 13.0. A value of p<0.05 was considered
statistically significant.
Gossypetin attenuate CP induced cytotoxicity
In order to evaluate the cytoprotective eect of gossypetin we
performed MTT cell viability assay. Human lymphocytes were
incubated with dierent gossypetin concentrations and 1 mM CP.
Figure 1 presents MTT assay results. At 24th hour, cells that were treated
with CP showed signicantly minimal cell viability rate when compared
to the control group (p<0.05). In addition, GP100 concentration has a
slight inhibitory eect on cell viability in human lymphocytes however
GP25 concentration increased cell viability. Cell viability was 57.75% in
CP group whereas in GP25, GP50 and GP100 was 130.74%, 86.20% and
81.89% respectively (p<0.05). When CP and dierent concentrations of
GP were applied on lymphocyte culture, they decreased the cytotoxic
eects of CP. Percentage of cell viabilities were 78.87% in CP+GP25,
79.74% in CP+GP50 and 81.89% in CP+GP100 group. MTT results
showed us all GP concentrations have protective role on CP toxicity
(Figure 1).
Flow cytometric analysis of Annexin V on human lymphocytes
e Annexin V binding assay is a universal, condential, user
friendly and fast ow cytometric technique that is used to discriminate
apoptotic and necrotic cell death, the cells were stained with Annexin
V-Cy3 and Sytotox Green, and the time course study was performed
using flow cytometry (Figure 2a and 2b). More than 39.9% of CP-
treated cells showed Annexin V-negative/Sytotox green-positive
staining at 2 h, and approximately 16.6% of the cells were Annexin
V-positive/Sytotox green-negative. When the cells were treated with
GP25 most of the cells remained double negative at 2 h, moreover,
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 90
GP25 increased the live cell number and decreased the number of
necrotic and late apoptotic cells, suggesting that GP25 did not induce
apoptosis at this concentration. In addition, 13.5% of CP+GP25 treated
cells showed Annexin V-negative/Sytotox green positive staining at 2
h, and approximately 9% of the cells were Annexin V-positive/Sytotox
green negative, and the percent of double negative cells were about
77%. is means that GP25+CP signicantly increased live cell number
and decreased necrotic and apoptotic cells when compared with the
CP treated group. GP50 treated cells showed 24% Annexin V-negative/
Sytotox green positive staining at 2 h, and approximately 15.2% of the
cells were Annexin V-positive/Sytotox green positive, and the rest 60%
were live cells. CP+GP50 treated group cells showed 21.9% Annexin
V-negative/Sytotox green-positive staining at 2 h, and approximately
25.4% of the cells were Annexin V-positive/Sytotox green negative. As a
result, GP50+CP combination increased apoptotic cells and decreased
necrotic cells when compared GP50 treated group. Last group GP100
treated cells showed 28.2% Annexin V-negative/Sytotox green positive
staining at 2 h, and approximately 16.4% of the cells were Annexin
V-positive/Sytotox green negative. When the cells were treated with
CP+GP100 combination, cells showed 36.9% Annexin V-negative/
Sytotox green positive staining at 2 h, and approximately 7% of the cells
were Annexin V-positive/Sytotox green negative. Our experimental
data showed that GP100+CP increased necrotic cell and decreased
apoptotic cells when compared with GP100 treated cells (Figure 3).
In summary, our ow cytometry assay results were complementary
to the MTT results; CP decreased the live cell number, on the other
hand GP25 increased the cell number near to the control group
levels. However, GP50 and GP100 decreased live cell number slightly.
Additionally, when we applied CP in combination with dierent
GP concentrations on human lymphocytes, both live cell numbers
increased and the percentages of apoptotic and necrotic cell decreased
signicantly when compared to the CP group. Also, our results
demonstrated that CP has necrotic and apoptotic eects on human
lymphocytes at 1 mM concentration. According to the data presented
in this article, the most eective concentration of GP was 25 µM against
CP toxicity (Figure 4).
Flow cytometric analysis of cell cycle proling with PI staining
Intranucleosomal DNA breaks are important markers of cellular
damage. eir presence means that apoptotic or necrotic common
Figure 1: Cell viability rate of human lymphocyte after exposure to CP, various
concentrations of GP and CP+GP combinations. ap<0.05 compare with control
group, bp<0.05 compare with CP group and cp<0.05 compare with G25 group.
Figure 2a and b: Analysis of living, apoptotic and necrotic cell numbers by
ow cytometer.
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 91
cascades takes place in the cell death arising from dierent causes. Cell
cycle proling with PI staining is a familiar, reliable, fast and easy ow
cytometric method for the detection of DNA breaks, cell cycle arrest
points and for the discrimination of viable, apoptotic or necrotic cells.
To determine whether the inhibitory eect of CP on human
lymphocyte cell proliferation involved cell cycle changes, we examined
cell cycle phase distribution of treated cells by ow cytometer. Our
ow cytometric analysis of cell cycle proling with PI staining revealed
complementary data to the MTT assay results. According to this study
results CP signicantly inhibited cell cycle in G0/G1 phase. On the
other hand, all of the applied gossypetin concentrations (GP25, GP50
and GP100) signicantly induced cell viability but G25 was the most
eective concentration among them. When GPs were applied with CP,
they decreased the blocking eect of CP on cell cycle. As a result, all of
the GPs have protected the cells against CP toxicity.
CP is an antineoplastic prodrug belongs to the nitrogen mustard
group which is converted to its active form in the liver. It is commonly
used in the treatment of autoimmune diseases and various cancer types
[16]. Although, CP takes place so oen in cancer therapy regimens, like
all other antineoplastic agents, may lead to new dysplasias which has the
possibility of emergence of new cancer types during treatment because
of its high toxic side eects. us, oncologists do not prefer to use CP
alone, instead of that they combine it with other drugs, for example
doxorubicin, to minimize the dosage for protecting normal healthy
cells from CP toxicity [17-19]. Besides this, scientists have improved
a new strategy against cytotoxic side eects of chemotherapeutic drugs
which is adding avonoids to the treatment regimens. erefore,
antineoplastic-immonosuppressive agents+avonoid combinations
are hot topic nowadays between researchers who are dealing with
cancer therapy or chronic progressive diseases. Xu et al. have worked
on ginsenoide Rg3 in combination with CP and revealed that this
drug+avonoid combination has prolonged the survival rate of mice
by eliminating the drug's toxic side eects, and inhibiting angiogenesis
via down regulating the VEGF (vascular endothelial growth factor)
expression [20]. In addition to that, Sammy et al. have combined CP
with luteolin and showed that their combination successfully decreased
the tumor volume, however, prevented the weight loss which might be
the sign of the healthy cell protection (one of the most common adverse
Figure 3: Percentages of live, necrotic and apoptotic cells in control and
experiment groups analyzed by ow cytometer.
Figure 4a and b: Flow cytometric analysis in cell cycle progression between
untreated and treated cultured human lymphocytes.
Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin against Cyclophosphamide Toxicity in Human Lymphocyte Culture In
vitro. Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
Med chem
ISSN: 2161-0444 Med chem, an open access journal Volume 6(2): 088-092 (2016) - 92
eects of chemotherapy) [21]. Furthermore, resveratrol+CP mixture
was tested in the treatment of breast cancer cells and found to be very
eective at inhibiting cell proliferation through arresting cell cycle and
leading them to the apoptosis [22]. Another study, which was designed
by Alkan et al. demonstrated that curcumin+CP combination arrested
breast cancer cell cycle at the G2/M phase, and led to cell cycle jamming
at S phase. Moreover, the combination of curcumin and CP upregulated
apoptotic Bax protein expression, however, downregulated anti-
apoptotic Bcl-2 protein expression. As a result, Bax/Bcl-2 balance was
changed in favor of apoptosis and breast cancer cells were directed to
the apoptosis by this way [23]. In the light of these ndings, we thought
that gossypetin too could be a good candidate for drug+avonoid
combinations strategy in cancer treatment for preserving healthy cells
against drug caused cellular damage because of its high ROS scavenging
capacity. Moreover, in this study, we have tested dierent GP and CP
concentrations on the human lymphocytes in vitro and observed cell
survival rates along with cell cycle proliferation phases. Consistent
with other publications in the literature [24] our study conrmed
that avonoids are capable of protecting normal healthy cells against
cytotoxic eects of chemotherapeutic agents like CP. According to our
results, GP has signicantly decreased cytotoxic eects of CP on human
lymphocytes. While all applied GP concentrations were successful
against CP damage, especially, the GP25 was the most eective dose
at reducing CP toxicity. Furthermore, our ow cytometric analysis of
cell cycle proling with PI staining was consistent with cell viability
results. CP arrested cell cycle in G0/G1 phase. On the other hand,
gossypetin addition to the culture media increased cell viability close
to the control group levels. Besides, GP25 increased cell viability via
suppressing apoptotic and necrotic impacts of CP. When GPs were
applied with CP, they decreased the blocking eect of CP on cell cycle.
As a result, all of the GPs have protected the cells against CP toxicity.
It means that, gossypetin ghts against CP derived cellular damage
through scavenging ROS, decreasing oxidative stress and supporting
antioxidant mechanisms. Finally, it is important to mention that GP is a
valuable and remarkable antioxidant biomolecule that could achieve to
be a good partner in cancer chemotherapy like luteolin and quercetin.
In conclusion, as far as we know this is the rst study in the literature
which demonstrates that GP has cytoprotective activity against CP
toxicity. e protective eects appear to be the result of the reduction
of cytotoxicity via inhibition of ROS production and restoration of the
antioxidant system. Although, we obtained promising results on the
lymphocyte cell culture experiments, further in vitro cancer cell culture
and animal studies are necessary for conrming whether the GP+CP
complex is ecient in physiological conditions to become a potential
new antitumour drug+avanoid combination.
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Citation: Ustunsoy S, Akal ZU, Alpsoy L (2016) Protective Role of Gossypetin
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Med chem 6: 088-092. doi:10.4172/2161-0444.1000330
... This is true despite the fact that pillararene-induced binding of environmentally relevant analytes has strong potential to facilitate such environmental remediation, provided that there is a mechanism to remove the analytes of interest after binding in the pillararene is achieved. Such environmentally relevant analytes that are focused on in this report include the polycyclic aromatic hydrocarbons (PAHs) anthracene, phenanthrene, and 1,10phenanthroline (33); the herbicide simazine (34) and the fungicide myclobutanil (35,36); the pharmaceutical drugs naproxen (37,38), carbamazepine (39,40), and cyclophosphamide (41,42), and the pharmaceutical metabolite clofibric acid (43,44); and the industrial chemicals dibutylphthalates (45,46) and perfluorooctanoic acid (PFOA) (47). ...
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The ability to bind and detect analytes with high levels of selectivity, sensitivity and broad applicability for a variety of analytes is an essential goal, with applications in public health and environmental remediation. Methods to achieve effective binding and detection include electrochemical, and spectroscopic methods. The use of supramolecular chemistry to accomplish such detection, by binding a target in a host and transducing that binding into a measurable signal, has advantages, including tunability of the sensor and the ability to rationally design hosts through an understanding of non-covalent interactions. Reported herein is the design and use of pillar[5]arenes to accomplish precisely such detection. Water-soluble pillar[5]arenes containing 10 cationic linker arms on their periphery bound toxicants in their hydrophobic cores with association constants of 10⁵–10⁶M⁻¹. With the use of cationic exchange resins, the pillar[5]arene hosts were removed from solution with their encapsulated guests, allowing for effective toxicant removal.
... We think that this may be due to the partial cytotoxic effect of high-dose apigenin on rat splenic lymphocytes [53]. In accordance with our results, Ustunsoy et al. [54] reported that gossypetin significantly enhanced the proliferation of human lymphocytes on cyclophosphamide treated conditions. Another study on the ethanolic extract of Cyrtomium macrophyllum (200 mg/kg) has been shown to potentially enhance the proliferation of splenic lymphocytes in mice, which was decreased by cyclophosphamide administration [55]. ...
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Aim: Major side effects of cyclophosphamide administration are immunosuppression and myelosuppression. The immunomodulatory effects of plant bioactive compounds on chemotherapy drug-induced immunosuppression may have significant effects in cancer treatment. For this reason, we investigated the immunomodulatory effect of myricetin, apigenin, and hesperidin in cyclophosphamide-induced immunosuppression in rats. Methods: In our study, a total of 64 rats were used, and divided into eight equal groups. These groups were: control, cyclophosphamide, cyclophosphamide + myricetin (100 mg/kg), cyclophosphamide + myricetin (200 mg/kg), cyclophosphamide + apigenin (100 mg/kg), cyclophosphamide + apigenin (200 mg/kg), cyclophosphamide + hesperidin (100 mg/kg), and cyclophosphamide + hesperidin (200 mg/kg). Myricetin, apigenin, and hesperidin pretreatments were performed for 14 d, while cyclophosphamide application (200 mg/kg) was performed only on the 4th day of the study. Levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, and natural killer cell cytotoxicity were determined. In addition, we measured pro-inflammatory cytokines, and followed lipid peroxidation and antioxidant markers and examined the histology of bone marrow, liver and spleen in all groups. Results: During cyclophosphamide treatment, all three phytochemicals increased the levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, antioxidant markers, and natural killer cell cytotoxicity. Moreover, the agents decreased the levels of pro-inflammatory cytokines and mediators, reduced lipid peroxidation markers, and reduced tissue damage in liver, spleen, and bone marrow. Conclusion: Our study demonstrated that myricetin, apigenin, and hesperidin can reduce the immunosuppressive effect of cyclophosphamide by enhancing both innate and adaptive immune responses, and these compounds may be useful immunomodulatory agents during cancer chemotherapy.
The present chapter provides an overview of the journey of food from hunger satisfaction to health- promoting agents. The inherent components of food such as carbohydrates, proteins, vitamins, lipids, antioxidants, and minerals are required for the normal physiological functioning of the body parts. In the modern era of the 21st century, the lifestyle and eating habits of people have been changed drastically. The change in eating habits and unhealthy diet leads to the emergence of modern diseases such as obesity, diabetes, hypertension, cancer, allergies, and cardiovascular diseases throughout the world. In view of the increase in life expectancy, side effects of modern-day drugs during their prolonged uses, the use of functional and nutraceutical food as a preventive step to control such diseases would be preferred. The present chapter highlights the role of foods and their bioactive components in human health, associated challenges, and future research perspectives. In addition, the role of science and technological applications especially waste-recycling approaches, nanotechnology, bioinformatics, toxicity prediction tools for the sustainable uses of functional and nutraceutical food, and the need for coordinated regulatory framework are discussed.
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In recent years, significant emphasis has been placed on combination chemotherapy in cancer using cytotoxic agents and plant derived-bioactive substances that are capable of selectively arresting cell growth and inducing apoptosis in tumour cells. The present study was undertaken to evaluate the possibility that the combination of curcumin and cyclophosphamide could show synergistic anti-proliferative effects towards CMT-U27 and CMT-U309 canine mammary cancer cells and, if so, to clarify the mechanism involved. The anti-proliferative activities of curcumin, cyclophosphamide and a combined treatment on CMT cells were determined using the MTT and LDH assays. The concentration of drug required for 50% inhibition of cell viability (IC50) and combination index (CI) values were calculated from log dose-response curves of fixed-combinations of curcumin and cyclophosphamide generated from MTT assays. Apoptosis was detected using a DNA fragmentation assay and Annexin-V/propidium iodide staining followed by flow cytometry. Cell cycle analyses were also performed using flow cytometry. The expression of the apoptosis-related proteins Bax and Bcl-2 was determined by immunocytochemical staining. MTT and LDH assays showed that curcumin and cyclophosphamide induced a dose-and a time-dependent decrease in cell viability. Isobole analysis revealed that the substances exhibited a synergistic interaction when IC50 and 1/2 IC50 concentrations of curcumin and cyclophosphamide were added concurrently to the cultures. This synergy was characterised by a significant increase in the percentage of early and late apoptotic CMT-U27 and CMT-U309 cells. However, internucleosomal fragmentation of DNA was not observed in the DNA fragmentation assay. Cells treated with curcumin and cyclophosphamide arrested at the G(2)/M and S phases of the cell cycle, respectively. In combined treatments cells were arrested in both phases of the cell cycle. Furthermore, immunocytochemical stainings demonstrated that the curcumin induced apoptosis in CMT cells by the modulation of Bcl-2/Bax protein expression, as the expression of Bcl-2 was decreased and that of Bax increased. This effect was more pronounced in combination treatments. In conclusion, our study shows that a combination of curcumin and cyclophosphamide shows synergistic growth inhibitory activity on CMT cells via induction of apoptosis and cell cycle arrest accompanied by modulation of Bcl-2/Bax protein expression. This finding provides a molecular basis for the development of natural compounds as novel anticancer agents and will allow lowering the dose of cytotoxic agents, which will in turn lead to more specific and less toxic therapies for mammary cancer in dogs.
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There has been a great advance in the pharmacognosy field, which has increased the use of medicinal plants by health professionals and practitioners of folk medicine. This systematic review explored the history and the present day application of phytotherapeutic medicines. We searched the databases Cochrane Library, Embase, Lilacs, PubMed, Scielo and a specialized bibliography. We concluded that there are many therapeutic potentialities in the use of phytotherapeutic medicines and medicinal plants. We also concluded that despite the increasing number of publications in the field each year, there is still a lack of reviews and meta-analyses that could promote a better integration of the knowledge produced. In addition, research about pharmacological interactions and multidisciplinary studies may promote a quicker and safer process from the workbench up to the clinical trials.
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Atherosclerosis is the process of hardening and narrowing the arteries. Atherosclerosis is generally associated with cardiovascular diseases such as strokes, heart attacks, and peripheral vascular diseases. Since the usage of the synthetic drug, statins, leads to various side effects, the plants flavonoids with antiartherosclerotic activity gained much attention and were proven to reduce the risk of atherosclerosis in vitro and in vivo based on different animal models. The flavonoids compounds also exhibit lipid lowering effects and anti-inflammatory and antiatherogenic properties. The future development of flavonoids-based drugs is believed to provide significant effects on atherosclerosis and its related diseases. This paper discusses the antiatherosclerotic effects of selected plant flavonoids such as quercetin, kaempferol, myricetin, rutin, naringenin, catechin, fisetin, and gossypetin.
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Abstract Cyclophosphamide (CTX), commonly used as an anti-neoplastic drug, can cause adverse side-effects including immunotoxicity and urotoxicity. Increasingly, plants have become sources of therapeutics that can help to restore host immunity to normal. In this study, Acacia ferruginea was assessed for an ability to protect mice against/mitigate CTX-induced toxicity. Co-administration of an extract of A. ferruginea (10 mg/kg BW, IP daily) for 10 consecutive days reduced CTX (25 mg/kg BW, IP daily)-induced toxicity. Apart from improvements in bladder and small intestine morphology, there was marked improvement in anti-oxidant (glutathione) levels in the bladder, suggesting a role for the anti-oxidant in reducing CTX-induced urotoxicity. Moreover, use of the extract significantly increased total leukocyte counts and bone marrow cellularity/α-esterase activity in CTX-treated mice which suggested a protective effect on the hematopoietic system. Co-treatment with the extract also prevented decreases in organ (liver, kidney, spleen, thymus) weight as well as body weight, thereby seemingly lessening the potential impact of CTX on the host immune system. Further, CTX-induced increases in serum aspartate transanimase, alanine transaminase, and alkaline phosphatase were reversed by extract co-treatment, as were alterations in in situ formation/release of interferon (IFN)-γ, interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α. Overall, this study indicated there were some protective effects from use of an extract of A.ferruginea against CTX-induced toxicities, in part through modulation of levels of anti-oxidants and pro-inflammatory cytokines.
Background: Chlorpyrifos (CP) is an organophosphorus pesticide that induces oxidative stress through the production of free radicals and depletes intracellular antioxidant reserves. In this study, the efficacy of three antioxidants (melatonin, coenzyme Q10 (CoQ10), and vinpocetine) on alleviation of toxic effects of CP was evaluated. Materials and methods: Cytotoxicity of CP, in the presence or absence of effective doses of melatonin, CoQ10, and vinpocetine, was determined in human peripheral blood lymphocytes after 72-h exposure. The levels of acetylcholinesterase (AChE) activity along with tumor necrosis factor α (TNF-α), as inflammatory index, were measured. Further, the viability and oxidative stress markers including cellular mitochondrial activity, cell death modes (apoptosis vs. necrosis), total antioxidant power (TAP), total thiol molecules (TTM), lipid peroxidation (LPO), and myeloperoxidase (MPO) activity were measured. Results: CoQ10 and also the combination of the three antioxidants were the most notable in opposing toxicity of CP and led to increasing TAP and TTM; improvement of AChE activity; and lowering LPO, MPO, TNF-α, and apoptosis compared to CP alone. Conclusion: CP toxicity overwhelms the intracellular antioxidant defense mechanisms. Exogenous supplementation with antioxidants, such as the ones we have investigated, seems to be effective in the prevention of cytotoxicity of CP.
More than half of all global deaths in 2010 were related to non-communicable diseases, including obesity, cancers, diabetes, and cardiovascular illnesses. It has been suggested that the alarming increase in the incidence of cardiovascular disease is the epidemiologic result of a nutrition transition characterized by dietary patterns featuring an increase in the intake of total fat, cholesterol, sugars, and other refined carbohydrates, concomitant with low consumption of polyunsaturated fatty acids and fiber. Although traditional dietary approaches have proven successful as part of the treatment for obesity and cardiometabolic derangements within clinical trial scenarios, they lack effectiveness in the long term, mainly due to poor compliance. Research has thus turned its attention to nutraceutics , nutrients that have the ability to modulate physiological and pathophysiological molecular mechanisms, thus resulting in favorable health outcomes. Polyphenols have been considered as among the bioactive molecules as they are thought to yield beneficial effects by exerting antioxidant activity; however, there are other - and even more robust - metabolic pathways through which polyphenols enhance cardiovascular health, such as via promoting vasodilatory, anti-atherogenic, antithrombotic, and anti-inflammatory effects. No standard dose has yet been determined, as the effects greatly vary among polyphenols and food sources; thus, there is an imperative need to generate more evidence in order to support dietary recommendations aimed at the prevention and therapeutics of obesity and its associated cardiometabolic diseases.
Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Gossypetin, a naturally occurring hexahydroxy flavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. In this study, the atheroprotective role of gossypetin was examined in endothelial cells. The protective effect of gossypetin against ox-LDL-induced injury in human umbilical vein endothelial cells (HUVECs) was first noted at 0.1-0.5 μM. Gossypetin showed potential in reducing the ox-LDL-dependent apoptosis as demonstrated by morphological and biochemical features, including apoptotic bodies formation, distribution of hypodiploid phase and caspase-3 activation. Next, the ox-LDL-induced formation of acidic vesicular organelles and the upregulation of the autophagy-related genes (LC3 and Beclin-1) were enhanced by gossypetin. The gossypetin-triggered autophagic flux was further confirmed by an increase of the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine (CQ). In addition, silencing of Beclin-1 inhibited both the gossypetin mediated protective affects and the autophagic process. Molecular data indicated that autophagic effect of gossypetin might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signalings, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA), and a PTEN inhibitor SF1670. Finally, gossypetin improved atherosclerotic lesions and endothelial injury in vivo. Our data imply that gossypetin upregulates the autophagic pathway, which led to subsequent reduction of ox-LDL-induced atherogenic endothelial cell injury and apoptosis, and provide a new mechanism for the antiatherosclerotic activity of gossypetin.
Gossypetin, a flavone originally isolated from Hibiscus species, has been shown to possess antioxidant, antimicrobial, and antimutagenic activities. Here, we investigated the mechanism(s) underlying the anti-atherosclerotic potential of gossypetin. 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay showed the addition of > 50 μM of gossypetin could scavenge over 50% of DPPH radicals. The inhibitory effects of gossypetin on the lipid and protein oxidation of LDL were defined by thiobarbituric acid relative substances (TBARS) assay, the relative electrophoretic mobility (REM) of oxidized LDL (ox-LDL), and fragmentation of apoB in the Cu(2+)-induced oxidation of LDL. Gossypetin showed potential in reducing ox-LDL-induced foam cell formation and intracellular lipid accumulation, and uptake ability of macrophages under non-cytotoxic concentrations. Molecular data showed that these influences of gossypetin might be mediated via peroxisome proliferator-activated receptor α (PPARα)/ liver-X receptor α (LXRα)/ ATP-binding cassette transporter A1 (ABCA1) and PPARγ/ scavenger receptor CD36 pathways, as demonstrated by the transfection of PPARα siRNA or PPARγ expression vector. Our data implied that gossypetin regulated the PPARs signals, which in turn led to stimulation of cholesterol removal from macrophages and delay atherosclerosis. These results suggested that gossypetin potentially could be developed as an anti-atherosclerotic agent.