Research Ideas and Outcomes : Research Idea
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Corresponding author: Raffaele Pezzani (raffaele.pezzani@unipd.it)
Received: 21 Jun 2016 | Published: 28 Jun 2016
© 2016 Raffaele Pezzani.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Pezzani R (2016) Saxifraga aizoides extract: novel potential effects on tumor cell models. Research Ideas and Outcomes 2: e9632. doi: 10.3897/rio.2.e9632
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Since ancient time nature has been used as the main source of herbal medicines. Even today many drugs derive or are elaborated from environmental flora. Of note natural products are fundamental sources of new anticancer drugs. Cancer is the second leading cause of death in the world and the fight against it still open and need every possible resources, including herbal product, which should be scientifically and deeply studied.
Saxifraga aizoides extract (SAE) has been never tested in preclinical tumor cell models.
Saxifraga aizoides, saxifragaceae, cancer cells, ethnopharmacology, phytotherapy
The use of herbal natural products is still the main source of medicines in many developing nations and is becoming increasingly popular among Western countries, especially over the past decades. Given the extraordinary interest about phytomedicine and ethnopharmacology, many researchers all over the world try to deepen and to understand the exact mechanism by which an herbal extract will interact with human body, and particularly with human diseases. In fact many successful uses of plant medicines have been derived from traditional/geographical use of plant, whose effects have been scientifically proved in recent years. In this context Traditional Chinese Medicine is gaining popularity and therapeutic efficacy, as numerous researchers (especially from China) scientifically investigated its role in human diseases (
Saxifraga is the largest genus in the family Saxifragaceae, containing about 440 species of holarctic perennial plants, known as saxifrages or rockfoils (
Saxifraga aizoides is a flowering herb of the genus Saxifraga, which lives in Europe and North America.
The aim of this project is to study the phytochemical profile of the ethanolic crude extract of Saxifraga aizoides (
This research project will try to discovery the novel effects of a Saxifraga aizoides crude extract (SAE). In particular it will explore the anti-proliferative role of SAE in 4 human cell models (ovarian, colon, prostate and breast cancer) and also in animal models (mice and rats) to evaluate anti-inflammatory and toxicity of SAE.
The research project aims at understanding the potential application of Saxifraga aizoides extract (SAE) on cancer cell models. In order to achieve this result, a scientific approach should be followed:
Materials and methods
Plant material
Saxifraga aizoides plant was collected in summer season 2015 at about 2000 meters above the sea level in Passo del Tonale (TN), Italy. Plant was identified according to Pignatti 1982. A voucher specimen number was deposited in the herbarium of the OU Endocrinology, University of Padova, with number 011SA. Plant was air-dried and powdered. 30g of powder was added to a mixture with ethanol (70%) for 24 h, then filtered. The solvent was removed by vacuum evaporator. A crude extract of Saxifraga aizoides was obtained (SAE).
GC/MS analysis of Saxifraga aizoides extract
The chemical analysis will be performed by re-solubilizing the dry extract in ethanol followed by gas-chromatography/mass spectrometry (GC/MS) analysis
Evalutation of anti-proliferative activity
Four different cell lines will be used: HeLa (ovarian cancer), HCT-116 (Colon cancer), PC-3 (Prostate cancer), MCF-7 (breast cancer) cell lines. Cell lines will be obtained by ATCC or will be a gift of other research labs. Cells will be cultured in DMEM/RPMI medium supplemented with 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. Cells will be plated on 96-well tissue culture microtiter plates at a density of 1x104 cells/well and treated with SAE, at different concentrations. MTT cell viability (Sigma-Aldrich) will be tested after 72 h of treatment, as described elsewhere. For each drug, we measured the Inhibitory Concentration 50 (IC50, defined as 50% of the inhibitory effect on cell viability). All experiments will be performed in quadruplicate and repeated three times.
The cytotoxic effects of SAE will be confirmed by the Trypan blue dye exclusion method. The assay will be performed at 72 h after treatment, using the single IC50 dose determined by MTT assay. At the end of treatment, cells will be collected by trypsinization, centrifuged and the cell pellet will be resuspended in 1 ml of PBS. Next, 10 ul of the resulting cell suspension will be admixed with 10 ul of Trypan blue (0.4% in PBS). The numbers of non-stained viable cells (NSt cells) and stained dead cells (St cells) will be counted using a hemocytometer. Cell viability will be then calculated by the following formula: Viability (%)= ¼ (NSt cells)/(St cells + NSt cells) x 100. Experiments will be performed in triplicated and repeated three times. The results will be interpreted as the ratio of viable cells after drug treatments to that of the untreated control.
Cell morphology assessment with Wright’s staining
Cells will be cultured on coverslips for 48h, incubated overnight in 0.1% FBS, and then treated with SAE for 72h. After that, cells will be washed in PBS, fixed in methanol for 5 min and Wright’s stained for 5 min. Cell morphology will be assessed by light microscopy at x400 magnification. The experiments will be repeated twice.
Statistical analysis
Statistical analysis will be performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA) and Microsoft Excel software. Cell line data will be analyzed using a two-tailed paired Student’s t-test. A p<0.05 will be considered statistically significant. Data will be expressed as mean ± standard error of mean (sem).
Expected results
We expect to obtain a SAE that will be used to explore its effects on cell cancer models. We will have a GC/MS profile of SAE. We will test the 4 cancer cell lines for viability after treatment with SAE, thanks to MTT and trypan blue assays. Furthermore cells morphology changes will be assessed by light microscopy. Clonogenic assay will be provide a specific insight on cell survival after treatment with SAE.
Implementation
The study of the Saxifraga aizoides extract (SAE) will be implemented by the in vivo experiments on animal models (both mice and rats).
Acute toxicity studies
The project will analyze the acute toxicity of the plant extract, evaluating the lethal dose (LD50) in mice. The method is described in
Anti-inflammatory studies
Anti-inflammatory study will be performed using wistar rats. The method is derived by
Ethics
The request to use animal models will be submitted to Local Ethics Committee only after successfully completion of in vitro studies. Procedures involving animals and their care will be in conformity with institutional guidelines that comply with national and international law and policies (D.L. 116/92 and subsequent implementing circulars), and experimental protocols will be approved by the Local Ethics Committee of Padua University (CEASA).
To be found.
University of Padova, Departament of Medicine DIMED, UO Endocrinology
Approved by Prof. Marco Boscaro, UO Endocrinology, DIMED, University of Padova, Italy
Insitutional Approval confirmation number: 01/2016 (date 2016-03-31)
Raffaele Pezzani: principal investigator.