Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and Additional Molecular Insights through Comprehensive Proteome Analysis via Nano LC-MS/MS.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α-bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated. OBJECTIVE: The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis. METHODS: The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis. RESULTS: The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses. CONCLUSION: The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.

Synergistic induction of apoptosis in lung cancer cells through co-delivery of PLGA phytol/α-bisabolol nanoparticles.

This study explored the potential of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles to enhance the effectiveness of anticancer treatments through combination therapy with phytol and α-bisabolol. The encapsulation efficiency of the nanoparticles was investigated, highlighting the role of ionic interactions between the drugs and the polymer. Characterization of PLGA-Phy+Bis nanoparticles was carried out using DLS with zeta potential and HR-TEM for size determination. Spectrophotometric measurements evaluated the encapsulation efficiency, loading efficiency, and in vitro drug release. FTIR analysis assessed the chemical interactions between PLGA and the drug actives, ensuring nanoparticle stability. GC-MS was employed to analyze the chemical composition of drug-loaded PLGA nanocarriers. Cytotoxicity was evaluated via the MTT assay, while Annexin V-FITC/PI staining and western blot analysis confirmed apoptotic cell death. Additionally, toxicity tests were performed on L-132 cells and in vivo zebrafish embryos. The study demonstrates high encapsulation efficiency of PLGA-Phy+Bis nanoparticles, which exhibit monodispersity and sizes of 189.3±5nm (DLS) and 268±54 nm (HR-TEM). Spectrophotometric analysis confirmed efficient drug encapsulation and release control. FTIR analysis revealed nanoparticle structural stability without chemical interactions. MTT assay results demonstrated the promising anticancer potential of all the three nanoparticle types (PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis) against lung cancer cells. Apoptosis was confirmed through Annexin V-FITC/PI staining and western blot analysis, which also revealed changes in Bax and Bcl-2 protein expression. Furthermore, the nanoparticles exhibited non-toxicity in L-132 cells and zebrafish embryo toxicity tests. PLGA-Phy+Bis nanoparticles exhibited efficient encapsulation, controlled release, and low toxicity. Apoptosis induction in A549 cells and non-toxicity in healthy cells highlight their clinical potential.

Anticancer effects of phytol against Sarcoma (S-180) and Human Leukemic (HL-60) cancer cells.

Phytol (Pyt), a diterpenoid, possesses many important bioactivities. This study evaluates the anticancer effects of Pyt on sarcoma 180 (S-180) and human leukemia (HL-60) cell lines. For this purpose, cells were treated with Pyt (4.72, 7.08, or 14.16 µM) and a cell viability assay was performed. Additionally, the alkaline comet assay and micronucleus test with cytokinesis were also performed using doxorubicin (6 µM) and hydrogen peroxide (10 mM) as positive controls and stressors, respectively. Results revealed that Pyt significantly reduced the viability and rate of division in S-180 and HL-60 cells with IC50 values of 18.98 ± 3.79 and 1.17 ± 0.34 µM, respectively. Pyt at 14.16 µM exerted aneugenic and/or clastogenic effects in S-180 and HL-60 cells, where the number of micronuclei and other nuclear abnormalities (e.g., nucleoplasmic bridges and nuclear buds) were frequently observed. Moreover, Pyt at all concentrations induced apoptosis and showed necrosis at 14.16 µM, suggesting its anticancer effects on the tested cancer cell lines. Taken together, Pyt showed promising anticancer effects, possibly through inducing apoptosis and necrosis mechanisms, and it exerted aneugenic and/or clastogenic effects on the S-180 and HL-60 cell lines.

Development of Galloyl Antioxidant for Dispersed and Bulk Oils through Incorporation of Branched Phytol Chain.

In this study, a novel galloyl phytol antioxidant was developed by incorporating the branched phytol chain with gallic acid through mild Steglich esterification. The evaluation of the radical scavenging activity, lipid oxidation in a liposomal model, and glycerol trioleate revealed its superior antioxidant activities in both dispersed and bulk oils. Then, the antioxidant capacity enhancement of galloyl phytol was further explored using thermal gravimetry/differential thermal analysis (TG/DTA), transmission electron microscopy (TEM), and molecular modeling. The EC50 values of GP, GPa, and GE were 0.256, 0.262, and 0.263 mM, respectively, which exhibited comparable DPPH scavenging activities. These investigations unveiled that the branched aliphatic chain enforced the coiled molecular conformation and the unsaturated double bond in the phytol portion further fixed the coiled conformation, which contributed to a diminished aggregation tendency and enhanced antioxidant activities in dispersed and bulk oils. The remarkable antioxidant performance of galloyl phytol suggested intriguing and non-toxic natural antioxidant applications in the food industry, such as effectively inhibiting the oxidation of oil and improvement of the quality and shelf life of the oil, which would contribute to the use of tea resources and extending the tea industry chain.

Butylated hydroxyl-toluene, 2,4-Di-tert-butylphenol, and phytol of Chlorella sp. protect the PC12 cell line against H2O2-induced neurotoxicity.

Oxidative stress is considered the main cause of cellular damage in a number of neurodegenerative disorders. One suitable ways to prevent cell damage is the use of the exogenous antioxidant capacity of natural products, such as microalgae. In the present study, four microalgae extracts, isolated from the Persian Gulf, were screened to analyze their potential antioxidant activity and free radical scavenging using ABTS, DPPH, and FRAP methods. The methanolic extracts (D1M) of green microalgae derived from Chlorella sp. exhibited potent free radical scavenging activity. In order to characterize microalgae species, microscopic observations and analysis of the expression of 18S rRNA were performed. The antioxidant and neuroprotective effects of D1M on H2O2-induced toxicity in PC12 cells were investigated. The results demonstrated that D1M significantly decreased the release of nitric oxide (NO), formation of intracellular reactive oxygen species (ROS), and the level of malondialdehyde (MDA), whereas it enhanced the content of glutathione (GSH), and activity of heme oxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), and catalase (CAT) in PC12 cells exposed to H2O2. The pretreatment of D1M improved cell viability as measured by the MTT assay and invert microscopy, reduced cell apoptosis as examined by flow cytometry analysis, increased mitochondrial membrane potential (MMP), and diminished caspase-3 activity. The GC/MS analysis revealed that D1M ingredients have powerful antioxidant and anti-inflammatory compounds, such as butylated hydroxytoluene (BHT), 2,4-di-tert-butyl-phenol (2,4-DTBP), and phytol. These results suggested that Chlorella sp. extracts have strong potential to be applied as neuroprotective agents, for the treatment of neurodegenerative disorders.

Phytol, a Constituent of Chlorophyll, Induces Root-Knot Nematode Resistance in Arabidopsis via the Ethylene Signaling Pathway.

Root-knot nematodes (RKNs; Meloidogyne spp.) parasitize the roots or stems of a wide range of plant species, resulting in severe damage to the parasitized plant. The phytohormone ethylene (ET) plays an important role in signal transduction pathways leading to resistance against RKNs. However, little is currently known about the induction mechanisms of ET-dependent RKN resistance. Inoculation of Arabidopsis thaliana roots with RKNs decreased chlorophyll contents in aerial parts of the plant. We observed accumulation of phytol, a constituent of chlorophyll and a precursor of tocopherols, in RKN-parasitized roots. Application of sclareol, a diterpene that has been shown to induce ET-dependent RKN resistance, to the roots of Arabidopsis plants increased phytol contents in roots accompanied by a decrease in chlorophyll in aerial parts. Exogenously applied phytol inhibited RKN penetration of roots without exhibiting nematicidal activity. This phytol-induced inhibition of RKN penetration was attenuated in the ET-insensitive Arabidopsis mutant ein2-1. Exogenously applied phytol enhanced the production of α-tocopherol and expression of VTE5, a gene involved in tocopherol production, in Arabidopsis roots. α-Tocopherol exerted induction of RKN resistance similar to that of phytol and showed increased accumulation in roots inoculated with RKNs. Furthermore, the Arabidopsis vte5 mutant displayed no inhibition of RKN penetration in response to phytol. These results suggest that exogenously applied phytol induces EIN2-dependent RKN resistance, possibly via tocopherol production.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytol Down-Regulates Expression of Some Cancer Stem Cell Markers and Decreases Side Population Proportion in Human Embryonic Carcinoma NCCIT Cells.

Cancer stem cells (CSCs), a subgroup of cancer cells, have self-renewal capacity and differentiation potential and drive tumor growth. CSCs are highly-resistant to conventional chemo-radio therapy. Phytochemicals were shown to be able to eliminate CSCs. Phytol is a diterpene alcohol with demonstrated anticancer effects. The current study compared the effect of phytol with retinoic acid (RA) as a well-known inducers of CSC differentiation and cisplatin, a common chemotherapy drug, on CSC markers in human embryonic carcinoma NCCIT cells. NCCIT cells were exposed to 10 mM RA for 14 day to induce differentiation. Moreover, NCCIT cells were treated with IC50 dose of cisplatin (12 µM) and phytol (40 µM) for 7 day. Real-time PCR showed that phytol was more effective that RA and cisplatin in down-regulating the CSC markers OCT4, NANOG, SOX2, ALDH1, ABCB1, CD44 and CD133. Percentage of SP (13%) and ABCB1+ (0.34%) in NCCIT cells decreased to 7% and 0.1% respectively after treatment with phytol. A very small proportion of NCCIT cells were positive for CD44 (0.2%) and CD133 (0.48%) and this fraction did not change significantly after treatment with three agents. In conclusion, phytol has the greatest inhibitory effect on CSC population and markers than RA and cisplatin.

Anti-diarrheal activities of phytol along with its possible mechanism of action through in-vivo and in-silico models.

Phytol (PHY), a chlorophyll-derived diterpenoid, exhibits numerous pharmacological properties, including antioxidant, antimicrobial, and anticancer activities. This study evaluates the anti-diarrheal effect of phytol (PHY) along with its possible mechanism of action through in-vivo and in-silico models. The effect of PHY was investigated on castor oil-induced diarrhea in Swiss mice by using prazosin, propranolol, loperamide, and nifedipine as standards with or without PHY. PHY at 50 mg/kg (p.o.) and all other standards exhibit significant (p < 0.05) anti-diarrheal effect in mice. The effect was prominent in the loperamide and propranolol groups. PHY co-treated with prazosin and propranolol was found to increase in latent periods along with a significant reduction in diarrheal section during the observation period than other individual or combined groups. Furthermore, molecular docking studies also suggested that PHY showed better interactions with the α- and ß-adrenergic receptors, especially with α-ADR1a and ß-ADR1. In the former case, PHY showed interaction with hydroxyl group of Ser192 at a distance of 2.91Å, while in the latter it showed hydrogen bond interactions with Thr170 and Lys297 with a distance of 2.65 and 2.72Å, respectively. PHY exerted significant anti-diarrheal effect in Swiss mice, possibly through blocking α- and ß-adrenergic receptors.

Phytol anti-inflammatory activity: Pre-clinical assessment and possible mechanism of action elucidation.

Phytol (PHY) is an acyclic natural diterpene alcohol and a chlorophyll constituent that exhibits several pharmacological effects, such as anticancer, antioxidant, and antimicrobial. Here, we aimed to assess the PHY anti-inflammatory effect in vitro and in vivo, and to deepen knowledge on the possible mechanism of action. For this purpose, egg albumin (in vitro) test was performed by using acetyl salicylic acid (ASA) as a standard nonsteroidal anti-inflammatory drugs (NSAID). For in vivo test, male Wistar albino rats were treated (intraperitoneally) with 100 mg/kg of PHY and/or standard NSAIDs ASA (100 mg/kg) and diclofenac sodium (Diclo-Na, 10 mg/kg) to evaluate the combined effect of PHY in formalin-induced paw edema model. Furthermore, an in silico (CADD) study was accomplished to assess the effect of PHY against cyclooxygenase (COX)-1 and 2 enzymes, nuclear factor kappa B (NF-κB), and interleukin-1ß (IL-1ß). Results revealed that PHY exhibits dose-dependent anti-inflammatory effect using the egg albumin method. PHY (100 mg/kg) co-treated with ASA and/or Diclo-Na reduced paw edema better than PHY alone or NSAIDs individual groups. Computational study showed that PHY efficiently interacts with COX-1 and 2, NF-κB, and IL-1ß. In conclusion, PHY exhibits anti-inflammatory activity, possibly via COX-1 and 2, NF-κB, and IL-1ß dependent pathways.

An osteoinductive effect of phytol on mouse mesenchymal stem cells (C3H10T1/2) towards osteoblasts.

In recent years, phytochemicals have been widely researched and utilized for the treatment of various medical conditions such as cancer, cardiovascular diseases, age-related problems and are also said to have bone regenerative effects. In this study, phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), an acyclic unsaturated diterpene alcohol and a secondary metabolite derived from aromatic plants was investigated for its effect on osteogenesis. Phytol was found to be nontoxic in mouse mesenchymal stem cells (C3H10T1/2). At the cellular level, phytol-treatment promoted osteoblast differentiation, as seen by the increased calcium deposits. At the molecular level, phytol-treatment stimulated the expression of Runx2 (a bone-related transcription factor) and other osteogenic marker genes. MicroRNAs (miRNAs) play an essential role in controlling bone metabolism by targeting genes at the post-transcriptional level. Upon phytol-treatment in C3H10T1/2 cells, mir-21a and Smad7 levels were increased and decreased, respectively. It was previously reported that mir-21a targets Smad7 (an antagonist of TGF-beta1 signaling) and thus, protects Runx2 from its degradation. Thus, based on our results, we suggest that phytol-treatment promoted osteoblast differentiation in C3H10T1/2 cells via Runx2 due to downregulation of Smad7 by mir-21a. Henceforth, phytol was identified to bolster osteoblast differentiation, which in turn may be used for bone regeneration.

Phytol, a Chlorophyll Component, Produces Antihyperalgesic, Anti-inflammatory, and Antiarthritic Effects: Possible NFκB Pathway Involvement and Reduced Levels of the Proinflammatory Cytokines TNF-α and IL-6.

Phytol is a diterpene constituent of chlorophyll and has been shown to have several pharmacological properties, particularly in relation to the management of painful inflammatory diseases. Arthritis is one of the most common of these inflammatory diseases, mainly affecting the synovial membrane, cartilage, and bone in joints. Proinflammatory cytokines, such as TNF-α and IL-6, and the NFκB signaling pathway play a pivotal role in arthritis. However, as the mechanisms of action of phytol and its ability to reduce the levels of these cytokines are poorly understood, we decided to investigate its pharmacological effects using a mouse model of complete Freund's adjuvant (CFA)-induced arthritis. Our results showed that phytol was able to inhibit joint swelling and hyperalgesia throughout the whole treatment period. Moreover, phytol reduced myeloperoxidase (MPO) activity and proinflammatory cytokine release in synovial fluid and decreased IL-6 production as well as the COX-2 immunocontent in the spinal cord. It also downregulated the p38MAPK and NFκB signaling pathways. Therefore, our findings demonstrated that phytol can be an innovative antiarthritic agent due to its capacity to attenuate inflammatory reactions in joints and the spinal cord, mainly through the modulation of mediators that are key to the establishment of arthritic pain.

Phytol loaded PLGA nanoparticles regulate the expression of Alzheimer's related genes and neuronal apoptosis against amyloid-ß induced toxicity in Neuro-2a cells and transgenic Caenorhabditis elegans.

Amyloid ß (Aß) induced neurotoxicity has been postulated to initiate synaptic loss and subsequent neuronal degeneration in Alzheimer's disease (AD). The nanoparticles based drug carrier system is considered as a promising therapeutic strategy to combat this incurable disease. It was also found to inhibit cholinesterase activity and apoptosis mediated cell death in Neuro-2a cells. The in vivo study further revealed that the Phytol and Phytol-PLGA NPs (Poly Lactic-co-Glycolic Acid Nanoparticles) was found to increase the lifespan, chemotaxis behavior and decrease Aß deposition & ROS (Reactive oxygen species) production in transgenic Caenorhabditis elegans models of AD (CL2006, CL4176). Phytol and Phytol-PLGA NPs treatment downregulated the expression of AD associated genes viz Aß, ace-1 and hsp-4 and upregulated the gene involved in the longevity to nematodes (dnj-14) and it also reduced the expression of Aß peptide at the protein level. Our results of in vitro and in vivo studies suggest that Phytol and Phytol-PLGA NPs hold promising neuroprotective efficacy and targets multiple neurotoxic mechanisms involved in the AD progression.

Phytol as an anticarcinogenic and antitumoral agent: An in vivo study in swiss mice with DMBA-Induced breast cancer.

Phytol (PHY) (3,7,11,15-tetramethylhexadec-2-en-1-ol) exhibits various pharmacological properties including toxicity and cytotoxicity, and exerts antitumor activity. Owing to the urgent need of new pharmaceutical formulations for breast cancer therapy, this study aimed at the evaluation of antitumor activity of PHY in 7,12-dimethylbenzanthracene-cancer-induced animal model. Comet assay was employed to evaluate the cytogenetics, DNA repair, and antigenotoxic activities of PHY in neoplastic (breast) and non-neoplastic rodent cells (bone marrow, lymphocytes, and liver). Additionally, hematological, biochemical, histopathological, and immunohistochemical analyses were carried out in experimental animals. Thirty nonpregnant female mice (n = 5) underwent 7 weeks treatment with 6 mg/kg pro-carcinogen, PHY (4 mg/kg), and cyclophosphamide (25 mg/kg). Induction of cancer was confirmed by histopathology and immunohistochemistry for Ki-67. Results suggest that PHY exhibits low toxicity in comparison with other groups in hematological, biochemical, histopathological, and organ size parameters. Additionally, PHY showed modulatory effects on the pro-carcinogen, and induced genotoxicity and apoptosis in breast cancer cells. Furthermore, it showed a DNA damage repair capacity in mouse lymphocytes. These data indicate that PHY may have the potential as an anticancer candidate in pharmaceutical consumption. © 2018 IUBMB Life, 71(1):200-212, 2019.

Phytol ameliorated benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice via inhibition of oxidative stress and apoptosis.

In the present study, the modulatory effect of phytol against benzo(a)pyrene [B(a)P] induced lung carcinogenesis was investigated in Swiss albino mice. During the experimental period, phytol treatment showed no adverse toxic effect and mortality to the experimental animals. Lung tumor was observed in B(a)P treated group and also in animals post-treated with low concentration (50 mg/kg) of phytol. No neoplastic changes were observed in the lung tissue of the animals treated with the maximum dose of phytol (100 mg/kg). An elevated level of antioxidant enzymes combined with macromolecular damage (lipid peroxidation, protein carbonyl content) was observed upon B(a)P treatment whereas, phytol restored the level of antioxidant enzymes which were comparable to the vehicle control group. Moreover, administration of B(a)P induced apoptosis, as observed by the highest expression of Bax, caspase-3, and caspase-9 proteins in lung tissue of B(a)P alone treated animals. However, phytol treatment reduced the expression of Bax, caspase-3, and caspase-9 protein and maintained the constant expression of anti-apoptotic protein Bcl-2. These observations positively reveal that phytol regulates the antioxidant enzymes and thereby protects the cells against B(a)P induced carcinogenesis without showing any adverse toxic effect to the animals.

Synergistic interaction of ß-caryophyllene with aromadendrene oxide 2 and phytol induces apoptosis on skin epidermoid cancer cells.

BACKGROUND: Pamburus missionis (Wight) Swingle (Rutaceae) is traditionally used in the treatment of swellings, chronic rheumatism, paralysis and puerperal diseases. In a previous study the authors demonstrated apoptotic activity of Pamburus missionis essential oil (EO) on A431 and HaCaT cells. The major components of EO were ß-caryophyllene (25.40%), 4(14),11- eudesmadiene (7.17%), aromadendrene oxide 2 (14.01%) (AO-(2) and phytol (6.88%). PURPOSE OF STUDY: To investigate the role as well as the interactions among EO components inducing apoptosis in A431 and HaCaT cells. METHODS: Isobolographic analysis and combination index methods were used to detect the type of interactions among the essential oil (EO) components. Cell viability was used to detect cytotoxic activity. Mechanism of cell death was studied using Annexin V-FITC/PI binding assay, cell cycle analysis, measurement of MMP and ROS generation by flow cytometry. Expression of apoptosis associated proteins was investigated by western blot. RESULTS: Combination of P. missionis EO components: ß-caryophyllene/ aromadendrene oxide 2 (ß-C/AO-(2)), ß-caryophyllene/phytol (ß-C/P) and aromadendrene oxide 2 /phytol (AO-(2)/P) inhibited growth and colony formation ability of skin epidermoid A431 and precancerous HaCaT cells. Synergistic interaction was observed between ß-C/AO-(2) and ß-C/P combination while AO-(2)/P exhibited an additive effect. Combination of components induced chromatin condensation, phosphatidylserine externalisation, increase in sub-G1 DNA content, cell cycle arrest at G0/G1 phase and intracellular ROS accumulation. Inhibition of intracellular ROS by N-acetyl cysteine treatment blocked apoptosis induced by the combinations. The combinations induced apoptosis in A431 and HaCaT cells mediated by loss of mitochondrial membrane potential (ΔΨm), increase in Bax/Bcl-2 ratio, release of cytosolic cytochrome c and activation of caspases (cleaved form of caspase-3, caspase-8, caspase-9) and by PARP cleavage. CONCLUSION: The present study demonstrates interactions among ß-C, AO-(2) and P in the induction of apoptosis on A431 and HaCaT cells. These data suggest the combination of ß-caryophyllene with aromadendrene oxide 2 and phytol could be potential therapeutics for the treatment of skin epidermoid cancer and precancerous cells.

Association of Phytol with Toxic and Cytotoxic Activities in an Antitumoral Perspective: A Meta-Analysis and Systemic Review.

BACKGROUND: Phytol have various pharmacological activities such as antimicrobial, cytotoxic, antitumoral, antimutagenic, anti-atherogenic, antidiabetic, lipid-lowering, antispasmodic, antiepileptic, antinociceptive, antioxidant, anti-inflammatory, anxiolytic, antidepressant and immunoadjuvant. Several studies point to an association of phytol with implications for apoptosis and necrosis at cellular levels in cancer, yet no clear conclusions were drawn. METHOD: To clarify this, we conducted a meta-analysis of non-clinical studies of phytol and its associations with toxicity and cytotoxicity emphasizing the mechanisms of apoptosis and necrosis induction and its importance in tumor therapy. Relevant studies were systematically searched in PubMed and Web of Science. The association between phytol and cyto-/toxicity was assessed by odds ratio (ORs) and 95% confidence intervals (CI). Twentythree studies were finally included in the meta-analysis. A significant association between phytol and toxicity (OR: 1.47; 95% CI = 0.86-2.48) was found among in vivo studies and cytotoxicity (OR: 1.81; 95% CI = 1.12- 2.65, p<0.05) in in vitro and ex vivo studies. In in vitro studies, 24% of them indicate that phytol at high doses induces apoptosis by several mechanisms; while about 40% of ex vivo studies indicate that phytol induces reactive oxygen species generation. But, Phytol does not act as a direct oxidant, unlike its metabolite phytanic acid. The 24% of in vivo studies also highlighted the mechanisms for apoptosis-like including expression of Bcl2 protein or mutations in pro-apoptotic protein Bax. Of them, 8% studies show necrosis and hepatotoxicity. However, in 24% of the articles, the mechanisms of toxicity and cytotoxicity are still not well elucidated. CONCLUSION: This study confirms that the association between phytol and cyto-/toxicity depends on the dose/concentration used in the given experimental conditions. Thus, there are still great prospects for new research aimed at the use of phytol and its metabolite as anticancer agents.

Phytol: A review of biomedical activities.

Phytol (PYT) is a diterpene member of the long-chain unsaturated acyclic alcohols. PYT and some of its derivatives, including phytanic acid (PA), exert a wide range of biological effects. PYT is a valuable essential oil (EO) used as a fragrance and a potential candidate for a broad range of applications in the pharmaceutical and biotechnological industry. There is ample evidence that PA may play a crucial role in the development of pathophysiological states. Focusing on PYT and some of its most relevant derivatives, here we present a systematic review of reported biological activities, along with their underlying mechanism of action. Recent investigations with PYT demonstrated anxiolytic, metabolism-modulating, cytotoxic, antioxidant, autophagy- and apoptosis-inducing, antinociceptive, anti-inflammatory, immune-modulating, and antimicrobial effects. PPARs- and NF-κB-mediated activities are also discussed as mechanisms responsible for some of the bioactivities of PYT. The overall goal of this review is to discuss recent findings pertaining to PYT biological activities and its possible applications.

Dietary phytol reduces clinical symptoms in experimental autoimmune encephalomyelitis (EAE) at least partially by modulating NOX2 expression.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. We investigated the effect of phytol in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), as phytol, a plant-derived diterpene alcohol, exerts anti-inflammatory and redox-protective actions. We observed a significant amelioration of clinical symptoms in EAE C57BL/6N mice fed prophylactically with a phytol-enriched diet. Demyelination, DNA damage, and infiltration of immune cells, specifically TH1 cells, into the central nervous system were reduced in phytol-fed EAE mice. Furthermore, phytol reduced T-cell proliferation ex vivo. Phytanic acid - a metabolite of phytol - also reduced T-cell proliferation, specifically that of TH1 cells. Additionally, phytol-enriched diet increased the mRNA expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 2 in white blood cells in the lymph nodes. Accordingly, phytol lost its anti-inflammatory effects in chimeric EAE C57BL/6N mice whose peripheral cells lack NOX2, indicating that phytol mediates its effects in peripheral cells via NOX2. Moreover, the effects of phytol on T-cell proliferation were also NOX2-dependent. In contrast, the T-cell subtype alterations and changes in proliferation induced by phytanic acid, the primary metabolite of phytol, were NOX2-independent. In conclusion, phytol supplementation of the diet leads to amelioration of EAE pathology in both a NOX2-dependent and a NOX2-independent manner via yet unknown mechanisms. KEY MESSAGES: Phytol diet ameliorates EAE pathology. Phytol diet reduces demyelination, immune cell infiltration, and T-cell proliferation. Phytol diet increases NOX2 mRNA expression in white blood cells in the lymph nodes. Phytol mediates its effects in peripheral cells via NOX2. Effects of phytol on T-cell proliferation were NOX2-dependent.

Phytol shows anti-angiogenic activity and induces apoptosis in A549 cells by depolarizing the mitochondrial membrane potential.

In the present study, the antiproliferative activity of phytol and its mechanism of action against human lung adenocarcinoma cell line A549 were studied in detail. Results showed that phytol exhibited potent antiproliferative activity against A549 cells in a dose and time-dependent manner with an IC50 value of 70.81 ±â€¯0.32 µM and 60.7 ±â€¯0.47 µM at 24 and 48 h, respectively. Phytol showed no adverse toxic effect in normal human lung cells (L-132), but mild toxic effect was observed when treated with maximum dose (67 and 84 µM). No membrane-damaging effect was evidenced by PI staining and SEM analysis. The results of mitochondrial membrane potential analysis, cell cycle analysis, FT-IR and Western blotting analysis clearly demonstrated the molecular mechanism of phytol as induction of apoptosis in A549 cells, as evidenced by formation of shrinked cell morphology with membrane blebbing, depolarization of mitochondrial membrane potential, increased cell population in the sub-G0 phase, band variation in the DNA and lipid region, downregulation of Bcl-2, upregulation of Bax and the activation of caspase-9 and -3. In addition, phytol inhibited the CAM vascular growth as evidenced by CAM assay, which positively suggests that phytol has anti-angiogenic potential. Taken together, these findings clearly demonstrate the mode of action by which phytol induces cell death in A549 lung adenocarcinoma cells.

Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer.