Citrus Peel in Maintaining Cellular Quiescence of Prostate Cancer

Cancer recurrence remains a major concern for cancer patients despite the significant advancement in cancer treatment. Cell cycle re-entry of quiescent cancer cells has been implicated as a key factor for cancer recurrence. The slow progression of prostate cancer allows a window of opportunity for intervention through diet. Therefore, the aim of this thesis was to evaluate the potential use of citrus peel extract in halting the cell cycle re-entry of quiescent prostate cancer cells. Firstly, water extract and hexane extract of citrus peel were prepared using a maceration technique. The anticancer activity of the extracts was evaluated on prostate cancer PC-3 and LNCaP cells using in-vitro assays. The cell viability analysis showed that hexane extract was more effective in reducing cell viability compared to water extract in both cell lines. For cell cycle analysis, quiescent prostate cancer PC-3 and LNCAP cells were released to re-enter cell cycle in the presence of extract. Water extract completely inhibited the entry of quiescent cells from G0/G1 phase to S phase upon release from quiescence. In contrast, the hexane extract completely allowed the cells progress to S and G2/M phase. However, both extracts significantly decreased the DNA synthesis rate in PC-3 cells compared to the control when analysed using EdU incorporation assay. Hesperidin and naringin, the predominant flavonoid compounds in water extract, exhibited negligible cell cycle re-entry inhibitory effect on quiescent PC-3 cells suggesting the contribution of other bioactive compounds in the extract. More importantly, the water extract showed no toxicity when tested on the normal human fibroblast cells. Subsequently, solvent extraction and chromatography techniques were used to identify the compounds in water extract that are responsible for the cell cycle re-entry inhibitory effect. The results showed that the water fraction exhibited the highest inhibitory effect among the fractions tested. Not only that, the inhibitory activity of fractions was lower than the water extract suggesting the compounds exhibited a greater effect in combination and the presence of a synergic effect. Using various analytical methods, citric acid was identified as one of the cell cycle re-entry inhibitor compounds present in water extract. The cell viability assay showed the IC50 of citric acid for prostate cancer PC-3 cells and normal epithelial prostate RWPE-1 cells were almost similar. However, citric acid significantly exhibited higher cell toxicity effect on PC-3 cells than RWPE-1 cells at a concentration above 1 mg/mL indicating the specific toxicity of citric acid on cancer cells. Not only that, the citric acid showed no significant toxicity effect on human normal fibroblast GM3348 cells when tested on similar concentration. As the mechanisms of cell cycle re-entry of quiescent cells remain largely unclear, a study was attempted to elucidate the protein changes by the citric acid at the cellular level. The proteomic experiment results revealed several proteins were regulated differently in the citric acid treated cells that were possible to act with the inhibition of cell cycle re-entry. The Ingenuity Pathway Analysis software predicted that citric acid possibly inhibited PC-3 cells from re-entering cell cycle mainly by suppressing PI3K/AKT and ERK/MAPK signalling pathways and activating PTEN pathway. Modulation of these signalling pathways by diet or drug has the potential to reduce the cell cycle re-entry of quiescent prostate cancer cells and preventing cancer recurrence. The outcomes of this study have widened the applications of citrus peel extract as a chemopreventive agent for post-therapy cancer patients. The recovery and utilization of bioactive compounds from citrus peel not only reduce the waste but also will open an avenue for the development of affordable fortifying food products with potential in reducing the risk of cancer recurrence

Combination of saponification and dispersive liquid–liquid microextraction for the determination of tocopherols and tocotrienols in cereals by reversed-phase high-performance liquid chromatography

A simple sample preparation technique coupled with reversed-phase high-performance liquid chromatography was developed for the determination of tocopherols and tocotrienols in cereals. The sample preparation procedure involved a small-scale hydrolysis of 0.5 g cereal sample by saponification, followed by the extraction and concentration of tocopherols and tocotrienols from saponified extract using dispersive liquid–liquid microextraction (DLLME). Parameters affecting the DLLME performance were optimized to achieve the highest extraction efficiency and the performance of the developed DLLME method was evaluated. Good linearity was observed over the range assayed (0.031–4.0 μg/mL) with regression coefficients greater than 0.9989 for all tocopherols and tocotrienols. Limits of detection and enrichment factors ranged from 0.01 to 0.11 μg/mL and 50 to 73, respectively. Intra- and inter-day precision were lower than 8.9% and the recoveries were around 85.5–116.6% for all tocopherols and tocotrienols. The developed DLLME method was successfully applied to cereals: rice, barley, oat, wheat, corn and millet. This new sample preparation approach represents an inexpensive, rapid, simple and precise sample cleanup and concentration method for the determination of tocopherols and tocotrienols in cereals

Combination of saponification and dispersive liquid–liquid microextraction for the determination of tocopherols and tocotrienols in cereals by reversed-phase high-performance liquid chromatography

A simple sample preparation technique coupled with reversed-phase high-performance liquid chromatography was developed for the determination of tocopherols and tocotrienols in cereals. The sample preparation procedure involved a small-scale hydrolysis of 0.5 g cereal sample by saponification, followed by the extraction and concentration of tocopherols and tocotrienols from saponified extract using dispersive liquid–liquid microextraction (DLLME). Parameters affecting the DLLME performance were optimized to achieve the highest extraction efficiency and the performance of the developed DLLME method was evaluated. Good linearity was observed over the range assayed (0.031–4.0 μg/mL) with regression coefficients greater than 0.9989 for all tocopherols and tocotrienols. Limits of detection and enrichment factors ranged from 0.01 to 0.11 μg/mL and 50 to 73, respectively. Intra- and inter-day precision were lower than 8.9% and the recoveries were around 85.5–116.6% for all tocopherols and tocotrienols. The developed DLLME method was successfully applied to cereals: rice, barley, oat, wheat, corn and millet. This new sample preparation approach represents an inexpensive, rapid, simple and precise sample cleanup and concentration method for the determination of tocopherols and tocotrienols in cereals

Citrus peel flavonoids as potential cancer prevention agents

Citrus fruit and in particular flavonoid compounds from citrus peel have been identified as agents with utility in the treatment of cancer. This review provides a background and overview regarding the compounds found within citrus peel with putative anticancer potential as well as the associated in vitro and in vivo studies. Historical studies have identified a number of cellular processes that can be modulated by citrus peel flavonoids including cell proliferation, cell cycle regulation, apoptosis, metastasis, and angiogenesis. More recently, molecular studies have started to elucidate the underlying cell signaling pathways that are responsible for the flavonoids' mechanism of action. These growing data support further research into the chemopreventative potential of citrus peel extracts, and purified flavonoids in particular. This critical review highlights new research in the field and synthesizes the pathways modulated by flavonoids and other polyphenolic compounds into a generalized schema

Rapid reversed-phase chromatographic method for determination of eight vitamin E isomers and gamma-oryzanols in rice bran and rice bran oil

Rice bran and rice bran oil contain high amounts of lipid soluble phytochemicals such as tocopherols, tocotrienols and γ-oryzanols with potential health benefits. A reversed-phase high-performance liquid chromatographic method for the quantitation of these phytochemicals was developed. These phytochemicals were extracted using rapid one-step methanol extraction and separated on a Kinetex pentafluorophenyl phase column for quantitation using fluorescence and ultraviolet detectors. Separation was carried out within 20 min at a flow rate of 1.0 mL/min using a gradient elution of mobile phase consisting of methanol and water at column temperature of 30 °C. A good separation of all eight tocol isomers was achieved while the γ-oryzanols were separated near to baseline. Extraction yields of tocopherols, tocotrienols and γ-oryzanols from saponification medium with different pH conditions were studied. Total γ-oryzanols extracted from alkaline medium were significantly lower than those from the neutral and acidic mediums (p  0.9994), low detection limit (3–34 ng/mL) and high precision (RSD < 7.5 %) with satisfactory recovery (R% = 81–116 %). The advantages of this method over available reverse-phase methods are the better resolution of β and γ tocol isomers with the γ-oryzanols, usage of non-halogenated mobile phase and shorter analysis time, and it is compatible with the standard laboratorial chromatographic equipment

Effect of addition of protein concentrates from natural and yeast fermented rice bran on the rheological and technological properties of wheat bread

The effect of substituting wheat flour with 0%, 5%, 10% and 15% protein concentrates from natural and yeast fermented rice bran on the rheological properties of their dough and bread properties was studied. Rheological properties of wheat dough were influenced by addition of rice bran protein concentrates. Overall acceptability score and specific loaf volume of 100% wheat bread were not significantly different from composite bread up to 10% rice bran protein substitution, and therefore, the optimised level of substitution was established. The optimised composite bread contained higher total amino acid content, radical scavenging activity and ferric reducing ability power (43.04–48.87 g/100 g, 182.77–201.65 mmol TEAC/100 g and 613.29–637.81 mmol TE/100 g) than control (33.86 g/100 g, 109.43 mmol TEAC/100 g and 540.13 mmol TE/100 g). Springiness, cohesiveness and resilience values of wheat bread were not significantly different from composite bread. Scanning electron microscopy revealed that composite bread had surfaces with embedded granules like protein deposits with small spores

Effect of citrus peel extracts on the cellular quiescence of prostate cancer cells

The re-entry of quiescent cancer cells to the cell cycle plays a key role in cancer recurrence, which can have a high risk after primary treatment. Citrus peel extracts (CPEs) contain compounds that can impair tumour growth, however the mechanism of action and effects on cell cycle regulation remain unclear. In this study, the capacity of ethyl acetate: hexane extract (CPE/hexane) and water extract (CPE/water) to modulate cell cycle re-entry of quiescent (PC-3 and LNCaP) prostate cancer cells was tested in an in vitro culture system. Cell cycle analysis showed that the quiescent PC-3 and LNCaP cancer cells in the presence of CPE/water were impaired in their ability to enter S phase where only 2-3% reduction of G0/G1 cells was noted compared to 12-18% reduction for control cells. In contrast, the CPE/hexane did not show any cell cycle inhibition activity in both cell lines. A low DNA synthesis rate and weak apoptosis were observed in quiescent cancer cells treated with CPEs. Hesperidin and narirutin, the predominant flavonoids found in citrus, were not responsible for the observed biological activity, implicating alternative bioactive compounds. Notably, citric acid was identified as one of the compounds present in CPE that acts as a cell cycle re-entry inhibitor. Citric acid exhibited a higher cell toxicity effect on PC-3 prostate cancer cells than non-cancerous RWPE-1 prostate cells, suggesting specific benefits for cancer treatment. In conclusion, CPE containing citric acid together with various bioactive compounds could be used as a chemopreventive agent for post-therapy cancer patients