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Projeto de investigação
Associated Laboratory for Green Chemistry - Clean Technologies and Processes
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Azadirachta indica A. Juss (neem) phenolic extract inhibits human B-lymphoblastoid cells growth via cell cycle arrest, apoptosis induction, and DNA damage
Publication . Santos, Klebson Silva; Costa, Carla; Bessa, Maria João; Teixeira, João Paulo; Muniz, Ana Veruska Cruz da Silva; Padilha, Francine Ferreira; Dariva, Cláudio; Oliveira, Maria Beatriz Pinto Prior
Aim:
As far as is known, the pharmaceutical effects of neem on human B-lymphoblastoid (TK6) cells have not been studied until now. Hence, the present study aimed to obtain neem phenolic extracts for inhibits the proliferation of TK6 cells and explore some possible underlying mechanisms involved in these effects.
Methods:
Hexane extract (HE) was obtained in the first step. After that, the residual hexane was removed from the neem. The dried neem sample was used in a new extraction for obtaining the ethyl acetate extract (EAE). Total phenolic compounds (TPC) and total flavonoid contents (TFC) were determined by spectrophotometric methods. Lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate the cytotoxicity in TK6 cells. The stop at G0/G1 cell cycle and inducing apoptosis in the TK6 cells were analyzed by flow cytometry. For deoxyribonucleic acid (DNA) damage evaluation, the alkaline comet test was used.
Results:
The higher TFC (65.50 mg/g of extract ± 1.17 mg/g of extract) and TPC (52.08 mg of extract ± 0.88 mg of extract) were obtained in EAE compared to HE that was obtained TFC of 14.61 mg/g of extract ± 0.60 mg/g of extract and TPC of 3.20 mg/g of extract ± 1.20 mg/g of extract. EAE was more significantly cytotoxic to TK6 cells than HE. The apoptosis induction was higher after exposure to 15.0 µg/mL of EAE (11.29%) in comparison to 15.0 µg/mL of HE (2.52%). The G0/G1 phase increased from 72% negative control (NC) to 83% after treatment with neem extracts (15 µg/mL). Neem extracts were also able to cause DNA strand breaks in TK6 cells.
Conclusions:
The extraction residue from neem leaf after hexane extraction is a source important of cytotoxic and genotoxic molecules against TK6 cells, the results also can suggest that the toxic effects in TK6 cells can be provided most likely due to the presence of high content of TPC from neem extracts.
Are chloropropanols and glycidyl fatty acid esters a matter of concern in palm oil?
Publication . Albuquerque, T.G.; Costa, H.S.; Silva, M.A.; Oliveira, M.B.P.P.
Background: Palm oil is the most consumed worldwide, being evident its importance due to several applications
by the food industry. The presence of toxic and mutagenic compounds, such as chloropropanols and glycidyl
fatty acid esters has increased the attention on this oil. Very recently, there have been several alerts about the
safety of certain foods containing palm oil in their composition. Consequently, the European Commission has
demanded the evaluation of these hazardous compounds in several food matrices, including palm oil, turning
this issue into a public health concern.
Scope and approach: This review focuses on chloropropanols and glycidyl fatty acid esters in palm oil, their
precursors, occurrence and mitigation strategies, as well as scientific opinions, current legislation and analytical
approaches.
Key findings and conclusions: Liquid chromatography coupled to mass spectrometry detection is preferred in the
analysis of chloropropanols and glycidyl esters in palm oil. Concerning the precursors, the presence of chlorides
and mono- and diglycerides is related to high amounts of these compounds, especially glycidyl esters. Several
differences in the occurrence of these compounds in palm oil were reported, namely in what concerns to applied
processing (refining/deodorization conditions; temperature/time of frying in the presence or absence of food).
There is still much work to be done to implement efficient mitigation strategies without compromising the safety
of palm oil. Furthermore, regarding the maximum levels in foodstuffs, European Commission is finalising the
new EU legislation which will include values for the maximum levels of glycidyl fatty acid esters in vegetable
oils.
Intestinal Permeability Studies using a more realistic barrier: performance of co-cultures of Caco-2/HT29-MTX cells
Publication . Figueira, Carolina; Gravato, Carlos; Alvito, Paula
The intestinal barrier, essential for overall health, can have its permeability affected by certain food compounds and additives. Among various models, in vitro cellular monolayers are the most commonly used to study this process. Among these, Caco-2 cells—representing enterocytes—are commonly used, though they lack complexity to mimic some properties of the intestinal barrier. This limitation can be overwhelmed by co-culturing it with HT29-MTX cells, which allows the secretion of mucus and mimics goblet cell functioning.
This study aimed to evaluate the intestinal permeability by assessing the paracellular and transcellular transport of lucifer yellow (LY) and propranolol (PR), two intestinal permeability markers, respectively, using a 9:1 co-culture of Caco-2/HT29-MTX cells.
Cells were cultivated separately in complete medium. Functional monolayer formation was monitored over 28 days using transepithelial electrical resistance (TEER) measurements in triplicate plates, with values ranging from 450.6 to 1287.3 Ω·cm². Transport assays were conducted on day 21 by applying LY and PR to the apical compartment and measuring their passage to the basolateral side. Apparent permeability coefficients (Papp) and basal recovery values were estimated by fluorescence quantification.
Statistical analyses were conducted to evaluate variability in TEER, Papp, and basal recovery results. The Shapiro-Wilk test was used to assess normality, and comparisons used one-way ANOVA followed by Tukey’s test or the Kruskal-Wallis test followed by Dunn’s test when requisites of ANOVA were not met. Significant differences in TEER values were observed between days 8–20 (p = 5.8e-11), 22–28 (p = 2.3e-09), and before vs. after transport on day 21 (p < 2.2e-16). For day 21 transport assays, a significant difference was found in LY basal recovery for two out of three plates (p = 0.03). As for the PR Papp values, there is a significant difference between duplicate plates (p = 0.005), as one plate was excluded due to a poor calibration curve fit.
These findings will inform improvements to the protocol for assessing intestinal permeability using co-culture models. Thus, Caco-2/HT29-MTX co-culture appears to be a promising model for evaluating the impact of food components and additives on the intestinal barrier.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UID/QUI/50006/2013