The effects of palm gamma-tocotrienol (GGT) on oxidative stress-induced cellular ageing was investigated in normal human skin fibroblast cell lines derived from different age groups; young (21-year-old, YF), middle (40-year-old, MF) and old (68-year-old, OF). Fibroblast cells were treated with gamma-tocotrienol for 24 hours before or after incubation with IC50 dose of H2O2 for 2 hours. Changes in cell viability, telomere length and telomerase activity were assessed using the MTS assay (Promega, USA), Southern blot analysis and telomere repeat amplification protocol respectively. Results showed that treatment with different concentrations of gamma-tocotrienol increased fibroblasts viability with optimum dose of 80 microM for YF and 40 microM for both MF and OF. At higher concentrations, gamma-tocotrienol treatment caused marked decrease in cell viability with IC50 value of 200 microM (YF), 300 microM (MF) and 100 microM (OF). Exposure to H2O2 decreased cell viability in dose dependent manner, shortened telomere length and reduced telomerase activity in all age groups. The IC50 of H2O2 was found to be; YF (700 microM), MF (400 microM) and OF (100 microM). Results showed that viability increased significantly (p < 0.05) when cells were treated with 80 microM and 40 microM gamma-tocotrienol prior or after H2O2-induced oxidative stress in all age groups. In YF and OF, pretreatment with gamma-tocotrienolprevented shortening of telomere length and reduction in telomerase activity. In MF, telomerase activity increased while no changes in telomere length was observed. However, post-treatment of gamma-tocotrienol did not exert any significant effects on telomere length and telomerase activity. Thus, these data suggest that gamma-tocotrienol protects against oxidative stress-induced cellular ageing by modulating the telomere length possibly via telomerase.
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A method for the determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with UV-vis detection has been developed. The separation of tocopherols was optimized in terms of mobile phase composition on the basis of the best compromise between efficiency, resolution, and analysis time. The optimal conditions were achieved using a C18 silica monolithic column (150 mm x 0.1 mm) with an isocratic elution of acetonitrile/methanol/water (acidified with 0.2% acetic acid) at a flow rate of 0.5 microL min(-1), giving a total analysis time below 18 min. The method has been applied to the quantification of tocopherols and tocotrienols present in several vegetable oils with different botanical origins.
Tumor regression after systemic administration of tocotrienol entrapped in tumor-targeted vesicles
Fu JY, Blatchford DR, Tetley L, Dufès C.
J Control Release. 2009 Dec 3;140(2):95-9. Epub 2009 Aug 23.
The therapeutic potential of tocotrienol, an extract of vitamin E with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. We hypothesize that the encapsulation of tocotrienol-rich fraction (TRF) within vesicles bearing transferrin, whose receptors are overexpressed on many cancer cells, could result in a selective delivery to tumors after intravenous administration. The objectives of this study are therefore to prepare and characterize transferrin-targeted vesicles encapsulating TRF, and to evaluate their therapeutic efficacy in vitro and in vivo. The entrapment of TRF in transferrin-bearing vesicles led to a 3-fold higher TRF uptake and more than 100-fold improved cytotoxicity in A431 (epidermoid carcinoma), T98G (glioblastoma) and A2780 (ovarian carcinoma) cell lines compared to TRF solution. The intravenous administration of TRF encapsulated in transferrin-bearing vesicles led to tumor regression and improvement of animal survival in a murine xenograft model, contrary to that observed with controls. The treatment was well tolerated by the animals. This work corresponds to the first preparation of a tumor-targeted delivery system able to encapsulate tocotrienol. Our findings show that TRF encapsulated in transferrin-bearing vesicles is a highly promising therapeutic system, leading to tumor regression after intravenous administration without visible toxicity.
BACKGROUND: Previous studies have shown that nicotine enhances oxidative DNA damage and leads to increased lipid peroxidation, which affects embryo development. The present study investigated the effect of daily supplementation of gamma-tocotrienol on oocytes of nicotine-treated mice.
MATERIAL/METHODS: Immature female mice (18-25 g) were divided into three groups. For 30 days, group A (control group) received saline (0.2 ml/day s.c.), group B nicotine (5 mg/kg/day s.c. in saline), and group C nicotine with gamma-tocotrienol (60 mg/kg/day p.o.). The animals were superovulated following these schedules.
RESULTS: Scanning electron microscopy (SEM) showed that the nicotine-treated oocytes appeared nonspherical with rough surface and the zona pellucida (zp) was torn and became irregular. Supplementation with gamma-tocotrienol in the nicotine-treated mice retained the spherical shape of the oocytes with intact zp; however, the surfaces of the oocytes remained irregular and rough. Transmission electron microscopy (TEM) following chronic nicotine treatment showed loosening of the boundary and tearing of the zp. The perivitelline space was also widened. The cytoplasm of the oocytes retained abundant rough endoplasmic reticulum (rER) with numerous vesicles. Mitochondria were highly dense, with no cristae. The administration of gamma-tocotrienol partially reduced the detrimental effects of nicotine by retaining the smooth boundary of the zp with the tight perivitelline space. There was less rER with no visible vesicle and a lower amount of dense mitochondrial matrix.
CONCLUSIONS: This study documented that chronic nicotine treatment adversely affects the ultrastructure of oocytes, while gamma-tocotrienol treatment at least minimizes the nicotine-induced damage to oocytes.
Modulation of melanin synthesis and its gene expression in skin melanocytes by palm tocotrienol rich fraction
Suzana Makpol, Nur Nadia Mohd Arifin, Zahariah Ismail, Chua Kien Hui, Yasmin Anum Mohd Yusof and Wan Zurinah Wan Ngah
African Journal of Biochemistry Research Vol.3 (12), pp. 385-392, December, 2009
Melanin is the pigment that determines skin color. Melanin synthesis is catalysed by the enzyme tyrosinase and is controlled by TYR, TYRP1 and TYRP2 genes. The objective of this study was to evaluate the anti pigmentation property of palm tocotrienol rich fraction by determining melanin synthesis and expression of genes involved in its regulation in skin melanocytes. Palm tocotrienol rich fraction (TRF) which contains 75% a-tocotrienol and 25% tocopherol was used to inhibit melanin synthesis which was determined by determining melanin level and tyrosinase enzyme activity. Expression of TYR, TYRP1 and TYRP2 genes was determined by quantitative real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Primary culture of skin melanocytes was divided into two groups; untreated control and cells that were treated with 500 μg/ml tocotrienol rich fraction for 24 h. Our results showed that there was a reduction in tyrosinase activity and melanin content in melanocytes treated with tocotrienol rich fraction compared to control (p < 0.05). Expression of TYRP2 gene in melanocytes treated with tocotrienol rich fraction was also decreased (p < 0.05) compared to control. In conclusion, palm tocotrienol rich fraction has an anti pigmentation property that inhibit melanin synthesis by inhibiting tyrosinase activity and down regulating TYRP2 gene expression.
The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats
Budin SB, Othman F, Louis SR, Bakar MA, Das S, Mohamed J.
Clinics (Sao Paulo). 2009;64(3):235-44.
Objective:This study examined the effects of palm oil tocotrienol-rich fractions on streptozotocin-induced diabetic rats.
Methods: Animals were divided into three groups: (i) normal non-diabetic (NDM), (ii) diabetic treated (tocotrienol-rich fractions – TRF) and (iii)diabetic untreated (non-TRF). The treatment group received oral administration of tocotrienol-rich fractions (200 mg/kg body weight) daily for eight weeks. The normal non-diabetic and the diabetic untreated groups were fed standard rat feed. Blood glucose and lipid profiles, oxidative stressmarkers and morphological changes of the thoracic aorta were evaluated.
Results: Tocotrienol-rich fractions treatment reduced serum glucose and glycated hemoglobin concentrations. The tocotrienol-rich fractions group also showed significantly lower levels of plasma total cholesterol, low-density lipoprotein cholesterol, and triglyceride, as compared to the untreated group. The tocotrienol-rich fractions group had higher levels of high-density lipoprotein cholesterol, as compared to the untreated group. Superoxide dismutase activity and levels of vitamin C in plasma were increased in tocotrienol-rich fractions-treated rats. The levels of plasma and aorta malondealdehyde + 4-hydroxynonenal (MDA + 4-HNE) and oxidative DNA damage were significant following tocotrienol-rich fractions treatment. Electron microscopic examination showed that the normal morphology of the thoracic aorta was disrupted in STZ-diabetic rats. Tocotrienol-richfractions supplementation resulted in a protective effect on the vessel wall.
Conclusion: These results show that tocotrienol-rich fractions lowers the blood glucose level and improves dyslipidemia. Levels of oxidative stressmarkers were also reduced by administration of tocotrienol-rich fractions. Vessel wall integrity was maintained due to the positive effects mediated bytocotrienol-rich fractions.
Attenuation of diabetic nephropathy by tocotrienol: Involvement of NFkB signaling pathway
Kuhad, A.,Chopra, K.
Life Sci, 2009;84(9-10);296-301.
Aim: Diabetic nephropathy is a serious complication for patients with diabetes mellitus. Approximately 30-40% of patients with type I and 15% with type II diabetes mellitus develop end stage renal disease. The study was designed to evaluate the impact of tocotrienol on renal function and reno-inflammatory cascade in streptozotocin-induced diabetes.
Main Methods: Streptozotocin (STZ)-induced diabetic rats were treated with tocotrienol (25, 50 and 100 mg/kg), alpha-tocopherol (100 mg/kg) or with vehicle form 5th to 8th weeks. After 8 weeks, urine albumin excretion, urine output, serum creatinine, blood urea nitrogen, creatinine and urea clearance were measured. Cytoplasmic and nuclear fractions of kidney was prepared for the quantification of oxidative-nitrosative stress (lipid peroxidation, superoxide dismutase, catalase, non protein thiols, total nitric oxide), tumor necrosis factor-alpha (TNF-alpha), tissue growth factor-1beta (TGF-beta1), p65 subunit of NFkappabeta and caspase-3.
Key Findings: After 8 weeks of STZ injection, the rats produced significant alteration in renal function, increased oxidative-nitrosative stress, TNF-alpha, TGF-beta1, caspase-3 activity in cytoplasmic lysate and active p65 subunit of NFkappabeta in nuclear lysate of kidney of diabetic rats. Interestingly, co-administration of tocotrienol significantly and dose-dependently prevented biochemical and molecular changes associated with diabetes. Tocotrienol (100 mg/kg) was demonstrated to be more effective than alpha-tocopherol (100 mg/kg). Moreover, diabetic rats treated with insulin-tocotrienol combination produced more pronounced effect on molecular parameters as compared to their respective groups.
Significance: Taken together, the data reveal that tocotrienol modulates the release of profibrotic cytokines, oxidative stress, ongoing chronic inflammation and apoptosis and thus exerts a marked renoprotective effect.
Tocotrienol attenuates oxidative-nitrosative stress and inflammatory cascade in experimental model of diabetic neuropathy
Kuhad, A.,Chopra, K.
Pharmacol Biochem Behav, 2009. 92(2):251-9.
Diabetic neuropathic pain, an important microvascular complication in diabetes mellitus, is recognised as one of the most difficult types of pain to treat. The development of tolerance, inadequate relief and potential toxicity of classical antinociceptives warrant the investigation of the newer agents to relieve this pain. Reactive oxygen/nitrogen species, cytokines and apoptosis are implicated in the pathogenesis of diabetic neuropathy. The aim of the present study was to explore the effect of tocotrienol on thermal and mechanical hyperalgesia, allodynia, oxidative-nitrosative stress, inflammation and apoptosis in streptozotocin-induced experimental diabetes. Diabetic rats developed neuropathy which was evident from a marked hyperalgesia and allodynia associated with enhanced nitrosative stress, release of inflammatory mediators (TNF-alpha, IL-1beta, TGF-1beta) and caspase-3. Chronic treatment with tocotrienol (25, 50 and 100 mg/kg body weight; p.o.) for 4 weeks starting from the 4th week of streptozotocin injection significantly attenuated behavioral, biochemical and molecular changes associated with diabetic neuropathy. Moreover, diabetic rats treated with insulin-tocotrienol combination produced more pronounced beneficial effect as compared to their per se groups. The major finding of the study is that insulin alone corrected the hyperglycemia and partially reversed the pain response in diabetic rats. However, combination with tocotrienol not only attenuated the diabetic condition but also reversed neuropathic pain through modulation of oxidative-nitrosative stress, inflammatory cytokine release and caspase-3 in the diabetic rats and thus it may find clinical application to treat neuropathic pain in the diabetic patients.
Evidence of gamma-tocotrienol as an apoptosis-inducing, invasion-suppressing, and chemotherapy drug-sensitizing agent in human melanoma cells
Chang PN, Yap WN, Lee DT, Ling MT, Wong YC, Yap YL.
Nutr Cancer. 2009;61(3):357-66.
To date, the most effective cure for metastatic melanoma remains the surgical resection of the primary tumor. Recently, tocotrienol-rich-fraction has shown antiproliferative effect on cancer cells. To elucidate this anticancer property in malignant melanoma, this study aimed, first, to identify the most potent isomer for eliminating melanoma cells and second to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of gamma-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of procaspases and the accumulation of sub-G1 cell population. Examination of the prosurvival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of NF-kappaB, EGF-R, and Id family proteins. Meanwhile, gamma-tocotrienol treatment also resulted in induction of JNK signaling pathway, and inhibition of JNK activity by selective inhibitor was able to partially block the effect of gamma-tocotrienol. Interestingly, gamma-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and gamma-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, synergistic effect was observed when cells were cotreated with gamma-tocotrienol and chemotherapy drugs. Together, our results demonstrated for the first time the anti-invasion and chemonsensitization effect of gamma-tocotrienol against human malignant melanoma cells.
Suppression of tumor growth by palm tocotrienols via the attenuation of angiogenesis
Weng-Yew W, Selvaduray KR, Ming CH, Nesaretnam K.
Nutr Cancer. 2009;61(3):367-73.
Previous studies have revealed that tocotrienol-rich fractions (TRF) from palm oil inhibit the proliferation and the growth of solid tumors. The anticancer activity of TRF is said to be caused by several mechanisms, one of which is antiangiogenesis. In this study, we looked at the antiangiogenic effects of TRF. In vitro investigations of the antiangiogenic activities of TRF, delta-tocotrienol (deltaT3), and alpha-tocopherol (alphaToc) were carried out in human umbilical vein endothelial cells (HUVEC). TRF and deltaT3 significantly inhibited cell proliferation from 4 microg/ml onward (P < 0.05). Cell migration was inhibited the most by deltaT3 at 12 microg/ml. Anti-angiogenic properties of TRF were carried out further in vivo using the chick embryo chorioallantoic membrane (CAM) assay and BALB/c mice model. TRF at 200 microg/ml reduced the vascular network on CAM. TRF treatment of 1 mg/mouse significantly reduced 4T1 tumor volume in BALB/c mice. TRF significantly reduced serum vascular endothelial growth factor (VEGF) level in BALB/c mice. In conclusion, this study showed that palm tocotrienols exhibit anti-angiogenic properties that may assist in tumor regression.