Moringa Oleifera aqueous leaf extract inhibits the growth of pancreatic cancer cell lines by down-regulating the NF-κB signaling pathway and increasing the cytotoxic effect of chemotherapy. The extract reduced the viability of three pancreatic cancer cell lines in a dose-dependent manner. It induced cell cycle arrest and apoptosis in Panc-1 cells by reducing levels of proteins involved in the NF-κB pathway. When combined with cisplatin chemotherapy, the extract synergistically enhanced the cytotoxic effect on Panc-1 cells. The study demonstrates that Moringa Oleifera extract can potentiate the efficacy of chemotherapy in pancreatic cancer by inhibiting the NF-κB pathway that contributes to chemoresistance.
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Comparative Cytotoxic Activities of the Flavonoid-Rich Ethyl Acetate Fruit Ex...inventionjournals
The fruit of Pouteriacampechianahas been previously reported to contain flavonoids and polyphenolic substances with high in vitro anti-oxidative effects. Its anti-tumorigenic activities have not been previously demonstrated. This study seeks to demonstrate the cytotoxic effects of the flavonoid-rich ethyl acetate fraction of the fruit extract of P. campechiana(EAFFPC) against K562 leukemic cell lines and healthy human whole blood cells. The standardized MTT-dye cell viability assay was carried out to measure the cytotoxicity of EAFFPC against the aforementioned cell lines cultured in RPMI. The assay showed that at a 60 µg well concentration, EAFFPC exhibited a 55.4% cell viability which is significantly lower than the cell viability obtained with 10 µg of vincristine. In contrast, EAFFPC demonstrated concentration-dependent cytoprotective properties in healthy human whole blood cells (HHWBC). This study confirms specific nonconcentration-dependent cytotoxic effects on K562 leukemic cell lines while exhibiting a concentrationdependent cytoprotective effects in HHWBC
This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.
Comparative Cytotoxic Activities of the Flavonoid-Rich Ethyl Acetate Fruit Ex...inventionjournals
The fruit of Pouteriacampechianahas been previously reported to contain flavonoids and polyphenolic substances with high in vitro anti-oxidative effects. Its anti-tumorigenic activities have not been previously demonstrated. This study seeks to demonstrate the cytotoxic effects of the flavonoid-rich ethyl acetate fraction of the fruit extract of P. campechiana(EAFFPC) against K562 leukemic cell lines and healthy human whole blood cells. The standardized MTT-dye cell viability assay was carried out to measure the cytotoxicity of EAFFPC against the aforementioned cell lines cultured in RPMI. The assay showed that at a 60 µg well concentration, EAFFPC exhibited a 55.4% cell viability which is significantly lower than the cell viability obtained with 10 µg of vincristine. In contrast, EAFFPC demonstrated concentration-dependent cytoprotective properties in healthy human whole blood cells (HHWBC). This study confirms specific nonconcentration-dependent cytotoxic effects on K562 leukemic cell lines while exhibiting a concentrationdependent cytoprotective effects in HHWBC
The current study investigated the immunomodulatory
potential of ethyl acetate soluble supernatant of
Lactobacillus casei (LC-EAS) in vitro. The effect of
LC-EAS on nitric oxide release was analyzed in RAW
264.7 cells, wherein, an inhibition in nitric oxide production
through suppression of inducible nitric oxide synthase
mRNA expression was observed. Evaluation of LC-EAS
on LPS-induced peripheral blood mononuclear cells
showed a down-regulation in TNF-a and IL-6 genes and an
upregulation of IL-10. An inhibition in the protein
expression of NF-kB, ERK1/2 and STAT3 phosphorylation
confirms the immunomodulatory potential of LC-EAS. The
effect of LC-EAS on in vitro intestinal epithelial cells was
investigated using HT-29 human colon adenocarcinoma
cancer cells. LC-EAS exhibited an inhibition of NF-jB and
ERK1/2 phosphorylation, whereas STAT3 phosphorylation
was unregulated. To evaluate the downstream target of
STAT3 upregulation, expression of the intestinal trefoil
factor TFF3 which is a NF-jB regulator and STAT3
downstream target was studied. LC-EAS was observed to
elevate TFF3 mRNA expression. Overall the study shows
that the anti-inflammatory potential of LC-EAS is through
inhibition of NF-kB in different cell types.
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
Deals with various methods adapted for the Improvement in Microbial Cell Culture alongwith the procedure and instruments used to carry out the operation through illustrative diagrams
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
The current study investigated the immunomodulatory
potential of ethyl acetate soluble supernatant of
Lactobacillus casei (LC-EAS) in vitro. The effect of
LC-EAS on nitric oxide release was analyzed in RAW
264.7 cells, wherein, an inhibition in nitric oxide production
through suppression of inducible nitric oxide synthase
mRNA expression was observed. Evaluation of LC-EAS
on LPS-induced peripheral blood mononuclear cells
showed a down-regulation in TNF-a and IL-6 genes and an
upregulation of IL-10. An inhibition in the protein
expression of NF-kB, ERK1/2 and STAT3 phosphorylation
confirms the immunomodulatory potential of LC-EAS. The
effect of LC-EAS on in vitro intestinal epithelial cells was
investigated using HT-29 human colon adenocarcinoma
cancer cells. LC-EAS exhibited an inhibition of NF-jB and
ERK1/2 phosphorylation, whereas STAT3 phosphorylation
was unregulated. To evaluate the downstream target of
STAT3 upregulation, expression of the intestinal trefoil
factor TFF3 which is a NF-jB regulator and STAT3
downstream target was studied. LC-EAS was observed to
elevate TFF3 mRNA expression. Overall the study shows
that the anti-inflammatory potential of LC-EAS is through
inhibition of NF-kB in different cell types.
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
Deals with various methods adapted for the Improvement in Microbial Cell Culture alongwith the procedure and instruments used to carry out the operation through illustrative diagrams
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
ABSTRACT- The anticancer drug arsenic trioxide is effective for acute promyelocytic leukemia. But the clinical trials are
restricted due to its potential side effects. Since the major part of arsenic metabolism and detoxification occurs in liver,
this organ faces the major threat. The hepatic side effects include fatty liver, fibrosis, and inflammation and hepatocyte
degeneration. Our study aimed to evaluate the protective potential of the fatty acid, docosahexaenoic acid, against adversities
of arsenic trioxide in an in vitro model, the Chang liver cells. Two preliminary dose standardization assays, cell
viability and lactate dehydrogenase release assays, were employed. The assays were performed as Pre-treatment,
Co-treatment and Post treatment experiments for a period of 24 hours. Arsenic trioxide at various doses (2.5, 5, 7.5, 10,
12.5 and 15 μM) showed a significant (p≤0.05) dose dependant reduction in cell viability along with a dose dependant
enhancement of lactate dehydrogenase release. However when the cells were treated with a combination of docosahexaenoic
acid at varying concentrations (50, 75, 100, 125 and 150 μM), the above mentioned conditions were found to be
reversed in Pre-treatment and Co-treatment experiments, but not in Post treatment. The most effective combination was
found to be 10 μM arsenic trioxide with 100 μM of docosahexaenoic acid in both Pre-treatment and Co- treatment studies.
Thus the preliminary assays of our study showed that docosahexaenoic acid administration as Pre-treatment or
Co-treatment can aid in reducing arsenic trioxide induced hepatotoxicity. Further studies are required to elucidate the mechanisms
behind the protective effects.
Key Words– Arsenic trioxide, hepatotoxicity, docosahexaenoic acid, cell damage
Propolis with its active component CAPE (Caffeic Acid Phenethyl Ester) stops breast cancer cell growth. These results of CAPE are present in the naturopathic formulation
of propolis, a widely available natural substance with an extended safety record, making it a naturally-occurring and readily available epigenetic agent with great potential in breast cancer and oncology in general. The ability to link the biological effects of a naturopathic remedy to the pharmacologic effects seen with an exciting class of drugs in the treatment of cancer opens the door to a host of new therapeutic opportunities for patients.
Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Ca...Jolene1981
ABSTRACT: The use of mushrooms contributes to human nutrition by providing low lipid content of lipids and high dietary fiber content, as well as significant content of other biologically active compounds such as polysaccharides, minerals, vitamins, and polyphenolic antioxidants. This study aimed to determine the content of polyphenols and polysaccharides, as well as the cytotoxic and antioxidative properties of several medicinal mushroom preparations. The content of total phenols and flavonoids of preparations of blended mushroom extracts (Lentifom, Super Polyporin, Agarikon, Agarikon Plus, Agarikon.1, and Mykoprotect.1) was evaluated quantitatively by using ultraviolet–visible spectroscopy spectrophotometric methods. The antioxidant capacity of the preparations was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and ferric reducing/antioxidant power assays. The content of water-soluble polysaccharides was determined using a specific gravimetric method, based on ethanol precipitation. To determine cytotoxic effects of single and blended mushroom extracts, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red assays were conducted using human small cell lung cancer, lung adenocarcinoma, colon cancer, and brain astrocytoma cancer cells. The obtained results suggest that due to the significant content of beneficial polyphenolic antioxidants and soluble polysaccharides, use of these mushroom preparations is beneficial in maintaining good health, as well as in the prevention and adjuvant biotherapy of various human pathological aberrations. These results reveal that these extracts exhibit different cytotoxic effects on tumor cells originating from different tissues. In addition, the comparison of investigated blended mushroom extracts with three well-known commercial mushroom products derived from single mushroom species or single mushroom compounds shows that blended mushroom extracts exhibit significantly stronger cytotoxic effects on human tumor cell lines.
Olive (Olea europaea) Leaf Extract and Chronic Myelogenous LeukemiaHakeem Zamano
Olive (Olea europaea) Leaf Extract Induces Apoptosis and
Monocyte/Macrophage Differentiation in Human Chronic
Myelogenous Leukemia K562 Cells: Insight into the Underlying
Mechanism
Indo-American Journal of Agricultural and Veterinary Sciences .It sounds like the journal you're referring to has a broad scope covering various aspects of Agricultural Sciences and Veterinary Medicine. The topics listed indicate a comprehensive range of fields within these discipline and submitting manuscripts to this journal can explore research and review articles of the journalism research.
The Indo-American Journal of Agricultural and Veterinary Sciences appears to be a scholarly journal focused on publishing research within the fields of agriculture and veterinary sciences of the journals public.
DOI:10.21276/ijlssr.2016.2.4.2
neoplastic progression through the action of viral oncoproteins, mainly E6 and E7.Cervical cancer remains the second
most common cancer in women worldwide with India as a major contributor to global burden with an annual incidence of
132,000 new cases and mortality rate of 74,000 deaths annually. In this study turmeric, neem, tulasi and ginger were
selected as natural anticancer drugs. The objective of the study was to analyze the anticancer property of turmeric
(Curcuma longa), neem (Azadirachta indica), tulasi (Occimum sanctum) and ginger (Zingiber officinale) on HeLa cells.
Turmeric, neem, tulasi and ginger capsules (Himalaya’s Company) were used and aqueous and methanolic extracts of the
turmeric, neem, tulasi and ginger were obtained using a soxhlet extraction. To check the efficacy of these drug MTT assay
was performed, that determines % viability and/or cytotoxicity. IC50 of aqueous turmeric, neem, tulasi and ginger extracts
in case of HeLa cells were 17.8, 22, 79.4, 27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger
extracts 17, 7.35, 75.24 and 16.1 respectively. To confirm apoptosis as the sole reason behind cell death
immunofluorescence based apoptosis assay was performed using TALI image based cytometer. The study has led to
postulate hypothesis that natural drugs e.g. turmeric, neem, tulasi and ginger are potent anti-cancer compound that are
capable of inhibiting the growth of immortal cells by apoptosis. Key-words- Cervical cancer, Human papillomavirus (HPV), Oncoproteins E6 and E7, Natural compounds, HeLa cell
line (adherent), Cell viability and MTT assay, Apoptosis assay
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Antioxidant and-anticancer-activities-of-moringa-leavesSilentdisco Berlin
Moringa is a plantfood of high nutritional value, ecologically and economically beneficial and readily available in the countries hardest hit by the food crisis. http://miracletrees.org/ http://moringatrees.org/
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
Despite considerable interest in the use of Moringa oleifera as a nutrient source, gaps and inconsistencies in the information on the nutrient content of this interesting plant remain. There are many reasons for this. The nutrient content of newly harvested plant material naturally varies with soil and climate as well as season and plant age. Differences in processing and storage procedures add more variation; and the use of different analytical techniques amplifies the variation further. For moringa leaves, additional variation has been created over time due to errors created as nutrient content values are incorrectly copied from source to source (30). The purpose of this review is to summarize the more recent scientific information about the nutrient content of fresh Moringa oleifera leaves and dried Moringa oleifera leaf powder. http://miracletrees.org/ http://moringatrees.org/
Moringa Oleifera, A Supermarket On A Tree
Moringa oleifera is extremely rich in vital nutrients and, as a bonus, can grow very fast in dry areas of the world, where food is scarce. Since ancient times, Moringa was used as a medicinal plant, known to heal and ease a wide number of diseases: from various inflammations to cancer, to parasitic diseases and diabetes. In more recent times, Moringa has gained notoriety as a nutrition power plant that can feed the needy and, in fact, save lives. And eyes… from blindness due to lack of vital nutrients such as vitamin A in the diet.
Moringa leaves or leaf powder can be used successfully as a suplement food to nourish small children, pregnant or nursing women, and of course, anybody else.
http://miracletrees.org/
2. synthesis using gemcitabine, with or without a second
agent like 5-FU or a platinum based agent [3]. Unfortu-
nately, this treatment is limited by a resistance of the
cancer cells to these therapies as well as a somatic tox-
icity [4]. The drug resistance is attributed to several
mechanisms: drugs exclusion from the cells, changes in
the enzymes metabolizing the drugs, or the cells becom-
ing more resistant to stress and apoptosis [4].
Nuclear factor kappa B (NF-κB) is an essential regula-
tor of the innate and adaptive immunity that, under
most conditions, supports proliferation and survival of
cells via inhibition of apoptosis [5]. Constitutively active
NF-κB signaling that strengthens the malignant cells’
ability to survive has been demonstrated in most tumor
cell lines as well as in a variety of patient-derived tumor
tissues, including those from pancreatic cancer [6,7].
NF-κB activation has also been directly linked to pancre-
atic cancer metastatic potential [8]. Although the reason
for its constitutive activation in malignant cells has not
been fully elucidated, suppression of NF-κB in the ma-
jority of these tumors leads to the induction of apoptosis
and the subsequent generation of cell death. In addition,
several lines of evidence indicate that NF-κB plays a sig-
nificant role in pancreatic cancer resistance to apoptosis-
based chemotherapies, leading to NF-kB being suggested
as a potential molecular target for pancreatic cancer
therapy [8].
Moringa Oleifera, Lam. (Moringaceae) is a tree that
grows widely in the tropics and subtropics of Asia and Af-
rica. Its leaves have been traditionally consumed by Asian
village people, but it is a relatively novel food material in
the western world [9]. Moringa Oleifera contains several
phytochemicals, some of which are of special interest be-
cause of their medicinal properties. Leaves of Moringa
Oleifera contain flavonoid pigments, such as kaempferol,
rhamnetin, isoquercitrin and kaempferitrin. In addition,
these leaves are rich in a group of the glycoside com-
pounds, glucosinolates and isothiocyanates [10] as well as
beta-sitosterol, glycerol-1-(9-octadecanoate), 3-O-(6'-
O-oleoyl-beta-D-glucopyranosyl), beta-sitosterol and beta-
sitosterol-3-O-beta-D-glucopyranoside, all of which have
demonstrated anti-cancer properties in-vitro [11]. An in-
vitro study using human KB cells as a cancer model has
shown that Moringa Oleifera leaf extract exerts strong
anti-tumor activity [12]. In addition, different leaf extracts
of Moringa Oleifera generate significant cytotoxic effects
on human multiple myeloma cultured cell lines [13].
The present study examined the effect of Moringa
Oleifera aqueous leaf extract on the viability and cell cycle
of cultured human pancreatic cancer cells, and evaluated
its ability to modify the expression of key proteins of the
NF-kB signaling pathway. Lastly, this study examined the
effect of combined treatments with Moringa Oleifera aque-
ous leaf extract and cisplatin chemotherapy on these cells.
Methods
Preparation of Moringa Oleifera aqueous extract
Leaves of Moringa Oleifera were received from Moringa
Arava Ltd, Israel. Moringa Arava grows the Moringa
Oleifera plant in the Dead Sea area, Israel, where it is
grown in rich mineral soil. The plant derived aqueous ex-
tract tested in this study was prepared in our laboratory
by mixing 1g dried and powdered leaves of Moringa
Oleifera with 10 mL boiling water for 5 minutes. The mix-
ture was then filtered twice through a 2 μm pore sterile fil-
ter paper into a sterile tube. The aqueous extract stock
solution (100 mg/mL) was freshly prepared for each set of
experiments and stored at 4°C for up to 5 days.
Cell lines and culture conditions
Human pancreatic cancer cell lines (Panc-1 and COLO-
357) were kindly provided by Prof. Ziv Gil (Laboratory
for Applied Cancer Research, Tel Aviv Sourasky Medical
Center, Israel). Human pancreatic cancer cell line p34
[14], developed from pleural effusion of a pancreatic
cancer patient, was kindly provided by Dr. Alex Starr
(Laboratory of Lung Biology, Lung and Allergy Institute,
Tel Aviv Sourasky Medical Center, Israel). All cells were
grown in Dulbecco modified Eagle medium (DMEM)
supplemented by 10% heat-inactivated bovine serum,
4.5 g/L glucose, 200 μM L-glutamine, 10 units/ml penicil-
lin and 10 μg/mL streptomycin (Biological Industries, Beit
HaEmek, Israel). The cells were incubated at 37°C in a 5%
CO2 humidified atmosphere. The cells were harvested by
trypsin/EDTA solution with 1–2 passages per week in a
split ratio of 1:3–5. The 24-hour cell cultures were used in
all the experiments.
Colorimetric tetrazolium salt (XTT) assay for cell survival
The effect of the treatments tested on the survival of
cultured cells was evaluated by XTT-based colorimetric
assay (Biological Industries). Typically, 200 μl with 1.5–
2x103
cells per well from exponentially growing cultures
were plated in 96 micro-well flat-bottom plates. After 24
hours had elapsed, the agents tested were added in cal-
culated concentrations each in three replicate wells and
incubated for 72 hours. At the end of the experiment, a
freshly prepared mixture of XTT and an activation re-
agent (PMS) was added into each well (50 μL). Following
2 hours of incubation at 37°C, the optical density (OD)
readings were measured at 450 nm using a microplate
reader (TECAN Sunrise™ , Switzerland). The measure-
ments were repeated following 3 and 4 hours of incuba-
tion, and the time point at which the assay showed
optimal OD readings was chosen to calculate the effect
of the treatment. When more than one time point fitted
these criteria, the results for the different time points
were normalized and averaged. Cell survival following
treatment was expressed as a percentage of viable cells
Berkovich et al. BMC Complementary and Alternative Medicine 2013, 13:212 Page 2 of 7
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3. relative to control value. All the experiments were
conducted at least twice, in triplicate wells each, and the
results of each experiment were averaged. The preliminary
experiments on the selected pancreatic cancer cell lines
had demonstrated that the OD readings correlated well
(r>0.97–0.99) with the number of seeded cells/well.
Analysis of synergistic effect of combined treatment
The IC50 value of each treatment was calculated on the
basis of dose–response curves produced by the XTT as-
says. In order to determine whether the combined treat-
ment is synergistic or additive, the data on cell survival
for each treatment alone and for combined treatment were
analyzed with Calcusyn software (Biosoft, Cambridge, UK)
that is based on the Chou and Talalay's equations [15]. This
software evaluates the combined effect of Moringa Oleifera
leaf extract and chemotherapeutic agents by calculating the
combination index (CI) using the following equation:
CI ¼
Dð Þ1
Dxð Þ1
þ
Dð Þ2
Dxð Þ2
Where (D)1 and (D)2 are the doses of drug 1 and drug
2 in a mixture that inhibits the system x percent and
where (Dx)1 and (Dx)2 are the doses of the drugs that
were given individually that inhibited the system x per-
cent. According to the software, CI ≤1 indicates syner-
gism, CI = 1 indicates an additive effect, and CI ≤1
indicates drug antagonism.
Immunoblotting analysis
The cells were plated in 10-cm tissue culture dishes for
24 hours at 37°C in a 5% CO2 humidified atmosphere.
The culture medium was then replaced with a fresh
medium supplemented with Moringa Oleifera leaf extract
for 24 hours. Whole-cell protein samples for immunoblot-
ting were prepared using a proteoJET™ mammalian cell lysis
reagent according to standard protocol (Fermentas Life Sci-
ences, Thermo Fisher Scientific). The cytoplasmic and nu-
clear extracts for immunoblotting were prepared using
NucBuster™ protein extraction kit (Novagen® ,U.S.A).
Determination of the protein concentrations was
performed using the Bradford assay. Protein from
each sample (100 μg) was subjected to electrophor-
esis in 10% SDS-PAGE and transferred to pure nitro-
cellulose blotting membranes (Millipore, Bedford,
M.A, U.S.A). Membranes were blocked for 1 hour at
room temperature with Tris (hydroxymethyl) amino
methane saline (TBS) containing 0.05% Tween 20
and 5% non-fat skim milk (BD Bioscience, San Jose,
CA). The membranes were then incubated at room
temperature in phosphate-buffered solution (PBS)
containing 5% milk and the following antibodies (1:200 di-
lutions): phospho-IκBα (Cell Signaling Technology®, M.A,
U.S.A), NF-κB, p65, IκBα and GAPDH (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). Membranes were
washed in TBS 0.05% Tween 20, and incubated with
either goat, anti-rabbit or rat secondary antibodies
(1:1000) conjugated to horseradish peroxidase (Santa
Cruz Biotechnology, Inc. U.S.A). Detection was by
SuperSignal® West Pico from Pierce BioLynx Inc.
(Brockville, Ontario, Canada) reagent.
Flow cytometry (FACS) analysis of cell cycle
The distribution of Panc-1 cells (intact and treated with
Moringa Oleifera leaf extract for 24 hours) in the cell
cycle was evaluated by flow cytometry. FACS analysis
was used to detect sub-diploid apoptotic cells, and try-
pan blue assay was used to discriminate between nec-
rotic and apoptotic cells. The cells (0.5-1.0x106
cells/
plate) were collected following treatment, washed with
cold PBS, fixed in ice cold 70% ethanol and kept at −20°C
for 24–48 hours. The pellets were washed in cold PBS
and suspended in PBS containing 0.1% Triton-X and
30 mg/mL DNAse-free RNAse A (Sigma-Aldrich, Israel)
for 6 hours at room temperature. Approximately one mi-
nute before the cells were analyzed, propidium iodide
(Sigma) in PBS was added at a final concentration of
10 μg/mL. The samples were analyzed using a FACSCallibur
instrument (BD Bioscience). Data were processed with BD
Bioscience software.
Statistical analysis
The results for each variant of treatment in these experi-
ments were represented as an average of 2–4 experi-
ments, and each arm was performed in triplicate. The
mean values and standard errors were calculated for
each time point from the pooled normalized data. The
statistical significance of the difference between groups
was determined by the two-tailed Student’s t-test. Values
of p < 0.05 were considered significant.
Results
The antiproliferative and apoptotic effects of the aque-
ous leaf extract of Moringa oleifera have been previously
evaluated in KB cultured human tumor cells [12], where
the extract was used in up to 200 μg/mL final concentra-
tion. Thus, for our survival experiments of cultured pan-
creatic cancer cells we started looking for an effect
around 0.1 mg/mL and upraised it according to our
results.
Effect of Moringa Oleifera leaf extract on survival and cell
cycle of pancreatic cancer cells
The effect of Moringa Oleifera leaf extract (0.1-2.0 mg/mL)
on the survival of cultured pancreatic cancer cells was eval-
uated following exposure for 72 hours. As shown in
Figure 1, Moringa extract inhibited the growth of all
Berkovich et al. BMC Complementary and Alternative Medicine 2013, 13:212 Page 3 of 7
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4. three tested cell lines. Panc-1 cells were more susceptible
to the treatment (IC50 = 1.1 mg/ml) compared to COLO
357 (IC50 =1.8 mg/ml) and p34 cells (IC50 = 1.5 mg/ml).
There was a significant inhibition of Panc-1 cell survival
at an extract concentration of 0.75 mg/mL. There was also
a significant inhibitory effect at a higher concentration
(1.5 mg/mL) in the two other cell lines. Moreover, treat-
ment with 2 mg/mL Moringa extract resulted in a 98%
reduction of Panc-1 cell survival.
We also evaluated the distribution of Panc-1 cells in the
cell cycle following 24 hours exposure to Moringa Oleifera
leaf extract using flow cytometry analysis of propidium
iodide-stained cells. It revealed a dose-dependent signifi-
cant increase of the percentage of cells in the sub-G1
phase, characterized by a very low DNA content, following
Moringa extract treatment (Figure 2A&B). This indicated a
fragmentation of the DNA as a result of progressive cell
apoptosis. Importantly, Moringa extract treatment also in-
duced significant (p < 0.05) apoptosis in Panc-1 cancer cells
at the minimal evaluated concentration of 0.25 mg/mL.
The induction of apoptosis was especially high (up to 30%)
at a concentration of 0.75 mg/mL.
Effect of Moringa Oleifera leaf extract on NF-κB signaling
pathway in pancreatic cancer cells
Moringa Oleifera leaf extract treatment of Panc-1 cells
down-regulated the expression of key NF-κB signaling
pathway proteins (Figure 3). The treatment of the cells
with 0.25 mg/mL extract for 24 hours resulted in a
down-regulation of p65, phospho-IκBα and IκBα pro-
teins levels compared to untreated cells (Figure 3A).
Treatments with 0.75 and 1.5 mg/mL Moringa extract
reduced or eliminated more significantly the presence of
all three proteins of the NF-κB signaling pathway. More-
over, p65 protein subunit levels have decreased in Panc-1
cells nuclei as a result of treatments with 0.1 - 1.5 mg/mL
Moringa extract. These data suggest that Moringa extract
attenuated pancreatic cancer cell’s survival ability, at least
in part, by targeting the NF-κB signaling pathway.
Combined effect of Moringa Oleifera leaf extract and
cisplatin
Based on the ability of Moringa extract to inhibit the
NF-κB signaling pathway, we hypothesized that its ex-
tract treatment would sensitize pancreatic cancer cells to
chemotherapy. We therefore tested the cytotoxic effect
of several combinations of Moringa Oleifera leaf extract
with cisplatin on Panc-1 cells. Since a synergistic effect
was expected, both the agents were used in the concen-
trations of a low inhibitory effect on proliferation of
Panc-1 cells. As shown in Figure 4, all the tested combi-
nations demonstrated an inhibitory effect higher than
the effects of each agent alone. The analysis of these data
by Calcusyn software, presented in Figure 4 and summa-
rized in the Table 1, clearly demonstrated that the com-
bined Moringa Oleifera leaf extract and cisplatin
regimen resulted in strong synergistic (CI = 0.1–0.3) or
synergistic (CI = 0.3–0.7) effects.
Discussion
The results of this study show that Moringa Oleifera leaf
extract can significantly inhibit the growth of cultured
human pancreatic carcinoma cells as well as its cell-
cycle progression, in a concentration-dependent manner.
Figure 1 Effect of Moringa Oleifera leaf extract on the viability of Panc-1, COLO 357 and p34 pancreatic cancer cells. The cells were
plated in 96-well plates and treated in triplicates with Moringa Oleifera leaf extract for 3 days. Cell survival was assessed using XTT-based cell
proliferation assay, and is indicated after normalization to the control group. The significance of the effect is indicated as *p ≤ 0.05 and
**p ≤ 0.001.
Berkovich et al. BMC Complementary and Alternative Medicine 2013, 13:212 Page 4 of 7
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5. Notably, the reduction of Panc-1 cells viability reached
100% following exposure to 2 mg/mL of Moringa extract
(Figure 1). To the best of our knowledge, this is the first
time that Moringa Oleifera was shown to have an effect
on pancreatic carcinoma cells. The exposure of Panc-1
cells to Moringa Oleifera leaf extract also reduced the
overall expression of key NF-κB family proteins in the
cells, as well as the levels of p65 protein subunit in the cell
nuclei (Figure 3). Consequently, this extract could inhibit
the NF-κB signaling cascade execution of target gene
transcription. Active NF-κB signaling was shown to
strengthen the pancreatic cancer cell’s ability to sur-
vive, and that suppression of NF-κB leads to induction
of apoptosis and thus generation of cell death [8]. In-
hibition of the NF-κB signaling cascade by Moringa ex-
tract explains, at least in part, its attenuating effect on
the survival of pancreatic cancer cells, as observed on
the viability assay (Figure 1).
Several lines of evidence indicate that NF-κB plays a sig-
nificant role in the resistance of pancreatic cancer to
apoptosis-based chemotherapies. Therefore, the NF-κB
signaling pathway was suggested as a potential molecular
target for combined therapy of pancreatic cancer [8,16].
Cisplatin is a platinum-based chemotherapeutic agent that
is known to have a minimal clinical efficacy in pancreatic
cancer due to tumor chemoresistance mechanisms [4]. It
Figure 3 Effect of Moringa Oleifera leaf extract on the expression of p65 NF-kB, IkB and p-IkB proteins in Panc-1. (A) Protein expressions
in crude lysates of the cells treated with Moringa Oleifera leaf extract (0–1.5 mg/mL) for 24 hours. (B) Expression of p65 in nuclear extracts of the
cells treated with Moringa Oleifera leaf extract. Protein expression was analyzed by Western blot. GAPDH and ß-actin were used to demonstrate
the quantity of standard proteins in the samples tested.
Figure 2 Effect of Moringa Oleifera leaf extract on the distribution of Panc-1 cells in the cell cycle. (A) Representative histograms of cell
distribution following Moringa Oleifera leaf extract treatments. (B) Data summarized from three independent experiments (%). The cells were
treated with the extract for 24 hours, collected, washed and fixed in 70% ice-cold ethanol. Prior to the analysis, the cells were suspended in 0.1%
Triton-X with 30 mg/ml DNAse-free RNAse A for 6 hrs. Propidium iodide was added several minutes before cell analysis by flow cytometry. The
significance of the effect, relatively to non-treated cells (control), is indicated as *p ≤ 0.05 and **p ≤ 0.001.
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6. has been shown in other human cancer models that in-
creased efficacy of cisplatin may be achieved by NF-κB in-
hibitors in cancer cells [17,18]. Therefore, we hypothesized
that the effect of Moringa Oleifera leaf extract on the in-
hibition of the NF-κB pathway may increase the efficacy of
cisplatin. As shown in Figure 4 and summarized in Table 1,
combined therapy of Moringa Oleifera leaf extract with cis-
platin demonstrated strong (CI = 0.1–0.3) or moderate
(CI = 0.3–0.7) synergistic effects in Panc-1 cells, re-
spectively. This indicates that the treatment of pancre-
atic cancer with cisplatin in combination with Moringa
Oleifera leaf extract may be a clinical therapeutic op-
tion for this chemoresistant cancer.
The past decade has witnessed the establishment of
herbal medicine as a rich source of new "western medicine"
drugs for multiple conditions, including cancer. These
herbs are often traditionally consumed by particular popu-
lations, presenting an advantage in terms of laying rest to
toxicity concerns. Herbs and their bioactive metabolites
have been reported to be anti-neoplastic both in ex-
perimental and clinical studies [19,20]. Moringa Oleifera
leaves contain flavonoid pigments, such as kaempferol,
rhamnetin, isoquercitrin and kaempferitrin. Flavonoid com-
pounds have various biological activities, including anti-
inflammatory and anti-cancer ones [21], and may be
mediating, at least in part, the effects shown here. In addition,
the Moringa Oleifera leaves are rich in a group of the glyco-
side compounds, glucosinolates and isothiocyanates [10], as
well as in beta-sitosterol, glycerol-1-(9-octadecanoate),
3-O-(6'-O-oleoyl-beta-D-glucopyranosyl), beta-sitosterol and
beta-sitosterol-3-O-beta-D-glucopyranoside, all of which
have demonstrated anti-cancer properties in-vitro [11] and
may have also contributed to the effects we exhibit. Since
this work does not include the isolation of bioactive
compounds from the Moringa Oleifera leaf extract, we
are not able to propose one or more specific active
compounds to be mediating the cellular anti-cancer ef-
fects demonstrated in our results. Moreover, it is pos-
sible that our findings can be attributed to additive or
Figure 4 Combined treatments effect of Moringa Oleifera leaf
extract with cisplatin on the viability of Panc-1 cells. (A) Cell
viability following treatments of Moringa Oleifera leaf extract,
cisplatin and combined treatments of increasing concentrations of
the extract and cisplatin for 3 days. (B) Normalized isobologram of
Moringa Oleifera leaf extract with cisplatin treatments shown in
compound A. (C) Median-effect plot curve of Moringa Oleifera leaf
extract, cisplatin and Moringa Oleifera leaf extract with cisplatin
shown in compound A. The cells were treated with each agent
alone or in combination for 3 days. Cell viability was evaluated using
XTT-based cell proliferation assay. Data was analyzed by
Calcusyn software.
Table 1 Calculated Combination Index (CI) of the effect of
combined treatments with Moringa Oleifera leaf extract
and cisplatin on Panc-1 cells viability
Combination index (CI) Cisplatin (μM) Moringa (mg/mL)
0.348 0.3 0.25
0.517 0.3 0.50
0.652 0.3 0.75
0.218 1.0 0.25
0.379 1.0 0.50
0.539 1.0 0.75
0.156 3.0 0.25
0.375 3.0 0.50
0.504 3.0 0.75
The cells were treated as described in Figure 4. Cell viability was evaluated
using XTT-based cell proliferation assay. Data was analyzed using
Calcusyn software.
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7. synergistic effects of several bioactive compounds from
the extract and not to a single one. Further research on
anti-cancer effects of specific bioactive metabolites derived
from Moringa Oleifere is warranted.
Toxicity has been a limiting factor in clinical pancre-
atic cancer treatment: combined therapy with cisplatin
and gemcitabine has shown promising results in phase II
and III trials, but it was followed by severe toxicity and
therefore has not been approved as a standard of care
[4]. This should not be the case regarding Moringa
Oleifera Lam, since an acute toxicity test carried out
using male Wistar albino mice, estimated the LD(50) of
orally administered aqueous leaf extract of Moringa
oleifera Lam. to be around 1585 mg/kg for a single ad-
ministration [22]. The aqueous leaf extract of Moringa
oleifera is, therefore, considered relatively safe when ad-
ministered orally and its acquisition of permission for
clinical use highly probable.
Still, future studies need to be conducted to evaluate
and quantify the therapeutic index of Moringa Oleifera
leaf extract applicable to pancreatic cancer patients in
the clinical setting.
Conclusions
Moringa Oleifera leaf extract inhibits the NF-κB signal-
ing pathway and increases the efficacy of chemotherapy
in human pancreatic cancer cells.
Abbreviations
NF-κB: Nuclear factor kappa-B; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide; CI: Combination Index; PBS: Phosphate-
buffered solution; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase;
OD: Optical density.
Competing interest
The authors declare that they have no competing interests.
Authors’ contributions
LB planned the study’s experiments, conducted the cell culture experiments,
analyzed the signaling assays and prepared the manuscript. SLA, AR and AV
performed the statistical analysis and data calculations. GE and IR assisted
with the manuscript. SLA conceived the study and gave final approval of the
manuscript. All authors read and approved the final manuscript.
Financial support
This study was supported by the Chaya and Kadish Shermeister Endowment.
Received: 21 February 2013 Accepted: 8 August 2013
Published: 19 August 2013
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doi:10.1186/1472-6882-13-212
Cite this article as: Berkovich et al.: Moringa Oleifera aqueous leaf
extract down-regulates nuclear factor-kappaB and increases
cytotoxic effect of chemotherapy in pancreatic cancer cells. BMC
Complementary and Alternative Medicine 2013 13:212.
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