1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated. 2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1-2 days. 3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1-3 days. 4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 microM tocotrienol at 1-3 days in culture compared to the control.
As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.
1. alpha-Tocopherol (alpha-T) and gamma-tocotrienol (gamma-T) were supplemented continuously for 8 weeks in the diets of normal rats and rats chemically induced with cancer using diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF) and partial hepatectomy. Hepatocarcinogenesis was followed by determining the plasma gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase (ALP) activities as well as placental glutathione S-transferase (PGST) and GGT activities histochemically, at 4-week intervals. 2. Male Rattus norvegicus were supplemented alpha-T and gamma-T at two different doses of 30 and 300 mg/kg diet. The supplementation was started at three different times: simultaneously with DEN administration; 4 weeks; and 8 weeks after DEN administration. 3. Elevation of plasma GGT activities and formation of PGST and GGT positive foci were attenuated significantly (P < 0.05) when alpha-T and gamma-T were supplemented simultaneously with cancer induction. Supplementation begun 4 and 8 weeks after cancer induction did not affect plasma enzyme activities and formation of enzyme-positive foci. 4. alpha-T was more effective than gamma-T, and a lower dose of 30 mg/kg was found to be more effective in reducing the severity of hepatocarcinogenesis.
The aim of this study was to determine the effects of gamma tocotrienol on lipid peroxidation and total antioxidant status of spontaneously hypertensive rats (SHR), comparing them with normal Wistar Kyoto (WKY) rats. SHR were divided into three groups and treated with different doses of gamma tocotrienol (gamma1, 15 mg/kg diet; gamma2, 30 mg/kg diet and gamma3, 150 mg/kg diet). Normal WKY and untreated SHR were used as normal (N) and hypertensive control (HC). Blood pressure were recorded every fortnightly for three months. At the end of the trial, animals were killed and measurement of plasma total antioxidant status, plasma superoxide dismutase (SOD) activity and lipid peroxide levels in plasma and blood vessels were carried out following well established methods. Study shows that lipid peroxides were significantly higher in hypertensive plasma and blood vessels compared to that of normal rats (Plasma- N: 0.06+/-0.01, HC: 0.13+/-0.008; p<0.001, B1. Vessels - N: 0.47+/-0.17, HC: 0.96+/-0.37; p<0.001). SOD activity was significantly lower in hypertensive than normal rats (N = 148.58+/-29.56 U/ml, HC = 110.08+/-14.36 U/ml; p = 0.014). After three months of antioxidant trial with gamma-tocotrienol, it was found that all the treated groups have reduced plasma lipid peroxides concentration but was only significant for group gamma1 (gamma1: 0.109+/-0.026, HC: 0.132+/-0.008; p = 0.034). On the other hand, lipid peroxides in blood vessels reduced significantly in all treated groups (gamma1; p<0.05, gamma2; p<0.001, gamma3; p<0.005). All the three treated groups showed improve total antioxidant status (p<0.001) significantly. SOD activity also showed significant improvement in all groups (gamma1: p<0.001, gamma2: p<0.05, gamma3: p<0.001). Correlation studies showed that, total antioxidant status (TAS) and SOD were significantly negatively correlated with blood pressure in normal rats (p = 0.007; p = 0.008) but not in SHR control. This correlation regained in all three groups SHR's after treatment with tocotrienol. Lipid peroxides in blood vessel and plasma showed a positive correlation with blood pressure in normal and SHR control. This correlation also remains in treated groups significantly except that in gamma3 where positive correlation with plasma lipid peroxide was not significant. In conclusion it was found that antioxidant supplement of gamma-tocotrienol may prevent development of increased blood pressure, reduce lipid peroxides in plasma and blood vessels and enhanced total antioxidant status including SOD activity.
We have investigated the pharmacokinetics and bioavailability of alpha-, gamma- and delta-tocotrienols under fed and fasted conditions in eight healthy volunteers. The volunteers were administered a single oral dose of mixed tocotrienols (300 mg) under fed or fasted conditions. The bioavailability of tocotrienols under the two conditions was compared using the parameters peak plasma concentration (Cmax), time to reach peak plasma concentration (Tmax) and total area under the plasma concentration-time curve (AUC(o-infinity)). A statistically significant difference was observed between the fed and fasted logarithmic transformed values of Cmax (P < 0.01) and AUC(0-infinity) (P < 0.01) for all three tocotrienols. In addition, the 90% confidence intervals for the ratio of the logarithmic transformed AUC(0-infinity) values of alpha-, gamma- and delta-tocotrienols under the fed state over those of the fasted state were found to lie between 2.24-3.40, 2.05-4.09 and 1.59-3.81, respectively, while those of the Cmax were between 2.28-4.39, 2.31-5.87 and 1.52-4.05, respectively. However, no statistically significant difference was observed between the fed and fasted Tmax values of the three homologues. The mean apparent elimination half-life (t(1/2)) of alpha-, gamma- and delta-tocotrienols was estimated to be 4.4, 4.3 and 2.3 h, respectively, being between 4.5- to 8.7-fold shorter than that reported for alpha-tocopherol. No statistically significant difference was observed between the fed and fasted t(1/2) values. The mean apparent volume of distribution (Vd/f) values under the fed state were significantly smaller than those of the fasted state, which could be attributed to increased absorption of the tocotrienols in the fed state.
The prevalence of diabetes is increasing worldwide. The World Health Organisation has estimated that there will be around 300 million diabetics by 2025. The largest increase will occur in Asia. The prevalence of type 2 diabetes is increasing due to a combination of factors: increasing lifespan, sedentary lifestyle, excessive intake of high energy foods, increasing prevalence of overweight/obese people. The Finnish Diabetes Prevention Study Group has clearly shown that changes in the lifestyle of both overweight men and women with impaired glucose tolerance can reduce the incidence of type 2 diabetes by 58%. This finding was confirmed by the Diabetes Prevention Programme which found that lifestyle intervention in individuals with impaired fasting glucose or impaired glucose tolerance reduced the risk of developing type 2 diabetes by 58%, whereas treatment with metformin reduced the risk of type 2 diabetes by only 31%. Both acarbose and troglitazone have also been shown to reduce the progression to diabetes in individuals who are at high risk of developing type 2 diabetes. Since the cure for diabetes remains some way off our concerted efforts should be directed at prevention of diabetes in order to curb the increasing prevalence of diabetes worldwide. Lifestyle changes are more beneficial than long term drug therapy in the prevention of diabetes and should be actively promoted.
A study was conducted to evaluate the bioavailability of alpha-, gamma- and delta-tocotrienols administered via oral, intravenous, intramuscular and intraperitoneal routes in rats. Three separate experiments, each conducted according to a two-way crossover design, were carried out to compare intravenous and oral, intramuscular and oral, and intraperitoneal and oral administration. Oral absorption of all three tocotrienols was found to be incomplete. Of the three tocotrienols, alpha-tocotrienol had the highest oral bioavailability, at about 27.7+/-9.2%, compared with gamma- and delta-tocotrienols, which had values of 9.1+/-2.4% and 8.5+/-3.5%, respectively. Such biodiscrimination was also observed in their total clearance rates (estimated from the intravenous data). alpha-Tocotrienol showed the lowest clearance rate at about 0.16 L kg(-1) h(-1), whereas that of delta- and gamma-tocotrienols was quite similar, with values of 0.24 and 0.23 L kg(-1) h(-1), respectively. Interestingly, all three tocotrienols were found to be negligibly absorbed when administered intraperitoneally and intramuscularly. Thus, these two routes of administration should be avoided when evaluating the biological activities of the tocotrienols in whole animal experiments.
A single dose comparative bioavailability study was conducted to evaluate the bioavailability of tocotrienols from two self-emulsifying formulations, one of which produced an emulsion that readily lipolysed under in vitro condition (SES-A), while the other produced a finer dispersion with negligible lipolysis (SES-B) in comparison with that of a non-self-emulsifying formulation in soya oil. The study was conducted according to a three-way crossover design using six healthy human volunteers. Statistically significant differences were observed between the logarithmic transformed peak plasma concentration (Cmax) and total area under the plasma concentration-time curve (AUC(0-infinity)) values of both SES-A and -B compared to NSES-C indicating that SES-A and -B achieved a higher extent of absorption compared to NSES-C. Moreover, the 90% confidence interval of the AUC(0-infinity) values of both SES-A and -B over those of NSES-C were between 2-3 suggesting an increase in bioavailability of about two-three times compared to NSES-C. Both SES-A and -B also achieved a faster onset of absorption. However, both SES-A and -B had comparable bioavailability, despite the fact that SES-B was able to form emulsions with smaller droplet size. Thus, it appeared that both droplet sizes as well as the rate and extent of lipolysis of the emulsion products formed were important for enhancing the bioavailability of the tocotrienols from the self-emulsifying systems.
Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H(2)O(2), a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H(2)O(2) for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H(2)O(2) induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.
Gamma-tocotrienol (GTT) has been shown to exhibit significant antitumor activity in a variety of tumor cells. Previous findings have demonstrated that GTT had antiprolifera-tive effects on a liver cancer cell line (HepG2) with an IC50 value of 170μM. In this study, two dimensional gel electrophoresis (2DE) was used to determine changes in protein expression in HepG2 cell line following treatment with GTT. The ultimate aim is to identify the possible molecular mechanisms involved in GTT antitumor activity. This study is focused on obtaining a 2DE protein profile for HepG2 cell line with and without
GTT treatment. In the preliminary analysis of the resulting 2DE profiles, 18 protein spots were found to be differentially expressed in cells treated with GTT. This observa-tion is confirmed by extending the analysis to a larger sample size. By studying the effects of GTT treatment on differential protein expression in HepG2 cells the underly-ing mechanisms involved in the antitumor activity of GTT may be elucidated.
This study was conducted to determine the effectiveness of three forms of vitamin E supplements following nicotine treatment on bone histomorphometric parameters in an adult male rat model. Rats were divided into seven groups: baseline (B, killed without treatment), control (C, normal saline for 4 months), nicotine (N, nicotine for 2 months), nicotine cessation (NC), tocotrienol-enhanced fraction (TEF), gamma-tocotrienol (GTT), and alpha-tocopherol (ATF). Treatments for the NC, TEF, GTT, and ATF groups were performed in two phases. For the first 2 months they were given nicotine (7 mg/kg), and for the following 2 months nicotine administration was stopped and treatments with respective vitamin E preparations (60 mg/kg) were commenced except for the NC group, which was allowed to recover without treatment. Rats in the N and NC groups had lower trabecular bone volume, mineral appositional rate (MAR), and bone formation rate (BFR/BS) and higher single labeled surface and osteoclast surface compared to the C group. Vitamin E treatment reversed these nicotine effects. Both the TEF and GTT groups, but not the ATF group, had a significantly higher trabecular thickness but lower eroded surface (ES/BS) than the C group. The tocotrienol-treated groups had lower ES/BS than the ATF group. The GTT group showed a significantly higher MAR and BFR/BS than the TEF and ATF groups. In conclusion, nicotine induced significant bone loss, while vitamin E supplements not only reversed the effects but also stimulated bone formation significantly above baseline values. Tocotrienol was shown to be slightly superior compared to tocopherol. Thus, vitamin E, especially GTT, may have therapeutic potential to repair bone damage caused by chronic smoking.
Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of alpha-tocopherol (alphaT) in PKC pathway and the anti-cancer properties of gamma-tocotrienol (gammaT3). This study aims to elucidate whether protective effects shown by alphaT and gammaT3 in H(2)O(2)-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H(2)O(2) is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of alphaT or gammaT3 at concentrations < or =10 microM. Similar to our previous work, gammaT3 was found to be neurotoxic at concentrations > or =100 microM, whereas alphaT showed no neurotoxicity. Cellular uptake of gammaT3 was higher than that of alphaT. Treating cells simultaneously with either gammaT3 or alphaT and with then H(2)O(2) led to higher expression of Bax and Bcl-2 than in neurons exposed to H(2)O(2) alone. Analysis of Bcl-2/Bax ratio as 'survival index' showed that both pretreatment of gammaT3 and alphaT followed by H(2)O(2) increase the 'survival index' of Bcl-2/Bax ratio compared to H(2)O(2)-treated cells, while treatment of gammaT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with alphaT alone. Similar treatment of gammaT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas alphaT did not alter its expression compared to H(2)O(2)-treated cells. Treating neurons with only gammaT3 or alphaT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with gammaT3 exerting stronger expression for proteins involved than alphaT. In conclusion, low doses of gammaT3 and alphaT confer neuroprotection to H(2)O(2)-treated neurons via their antioxidant mechanism but gammaT3 has stronger pro-apoptosis tendency than alphaT by activating molecules involved in the neuronal apoptotic pathway in the absence of H(2)O(2).
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.
Recently, vitamin E has been found to promote the bone structure of nicotine-treated rats well above their baseline values, thus suggesting that vitamin E may have some anabolic action. A bone anabolic agent acts by improving the bone structure leading to stronger bone. To assess the possible anabolic action vitamin E on bone, we supplemented alpha-tocopherol (ATF) or gamma-tocotrienol (GTT) at 60 mg/kg or vehicle [normal control (NC) group] for 4 months to normal male rats and measured their bone structure and biomechanical properties. Histomorphometric analysis revealed that vitamin E-supplemented rats have better trabecular volume, thickness, number, and separation than rats receiving vehicle only. For the first time we reported that GTT improves all the parameters of bone biomechanical strength, while ATF only improved some of the parameters compared to the NC group. Vitamin E supplementation, especially with the gamma isomer, improves bone structure, which contributed to stronger bone. Therefore, vitamin E has the potential to be used as an anabolic agent to treat osteoporosis or as bone supplements for young adults to prevent osteoporosis in later years.
Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and gamma-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.
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-tocotrienol prevented 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.
Intake of the antioxidant vitamins C and E lowers the oxidative stress. The study aimed to determine plasma concentrations of vitamin C and tocotrienols after supplementation of both vitamins in young male adults.
Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (p
Oxidative stress is characterised by an increased level of reactive oxygen species (ROS) that disrupts the intracellular reduction-oxidation (redox) balance and has been implicated in various diseases including cancer. Malignant tumors of connective tissue or sarcomas account for approximately 1% of all cancer diagnoses in adults and around 15% of paediatric malignancies per annum. There exists no information on the alterations of oxidant/antioxidant status of sarcoma patients in literature. This study was aimed to determine the levels of oxidative stress and antioxidant defence in patients with primary bone and soft tissue sarcoma and to investigate if there exists any significant differences in these levels between both the sarcomas.
Sisa puri pitaya merah bersama biji telah digunakan dalam penyediaan mufin yang dicampurkan dengan 10%, 15% dan 20% puri dalam formulasi bater. Ujian warna, kandungan jumlah polifenol, jumlah flavonoid, ujian antioksida; pemerangkapan radikal bebas 2,2-difenil-1-pikrilhidrazil (DPPH) dan ujian penurunan ferrik (FRAP) telah dijalankan. Warna bater 3 jenis mufin tersebut berwarna merah jambu violet. Warna merah jambu (nilai a) meningkat dengan signifikan (p<0.05) selaras peningkatan peratus kandungan puri manakala kecerahan (nilai L) menurun dengan peningkatan puri. Apabila dimasak, semua warna merah jambu daripada bater hilang. Permukaan mufin adalah lebih gelap (nilai L), dengan mufin 20% puri paling signifikan (p<0.05). Isi kesemua mufin berwarna kuning dan kecerahan (nilai L) didapati berkurang secara signifikan (p<0.05) dengan pertambahan peratus puri. Jumlah polifenol sampel mufin menunjukkan mufin 10% puri pitaya ekstrak air mengandungi jumlah polifenol yang paling tinggi (29.0 mg GAE/100 g sampel). Kandungan flavonoid menunjukkan mufin 20% puri pitaya mengandungi flavonoid yang paling signifikan (p<0.05) 15.3 mg katekin/100 g sampel berbanding mufin kawalan 11.0 mg katekin. Bagi ujian antioksida DPPH, semua mufin dengan puri pitaya menunjukkan peratus pemerangkapan yang lebih baik berbanding kawalan. Ujian FRAP menunjukkan pola yang serupa dengan keputusan mufin 10% (17.4), mufin 15% (15.4) dan mufin 20% (17.5 mg trolox/100 g sampel). Warna merah jambu bater mufin hilang semasa proses memasak namun nilai antioksida masih diperolehi dalam mufin masak.