A Fast and Efficient Ultrasound-Assisted Extraction of Tocopherols in Cow Milk Followed by HPLC Determination.

A fast HPLC method with fluorescence detector (FD) was developed for the determination of three tocopherols (TOCs) in milk samples from Modicana cattle breed. The ultrasound-assisted procedure was optimized for the extraction of TOCs prior to HPLC/FD analysis, reducing sample preparation time and allowing a fast quantification of α-tocopherol, δ-tocopherol and γ tocopherol. The optimized ultrasonic extraction combines an efficient and simple saponification at room temperature and a rapid HPLC quantification of TOCs in milk. The precision of the full analytical procedure was satisfactory and the recoveries at three spiked levels were between 95.3% and 87.8%. The linear correlations were evaluated (R2 > 0.99) and the relative standard deviation (RSD) values for intra-day and inter-day tests at three spiked levels were below 1% for the retention time and below 5.20% for the area at low level spiking. The proposed procedure, reducing the experimental complexity, allowed accurate extraction and detection of three TOCs in milk samples from Modicana cattle breed.

Whey protein isolate-gelatin nanoparticles enable the water-dispersibility and potentialize the antioxidant activity of quinoa oil (Chenopodium quinoa).

The quinoa oil presents benefits to health, but its low water dispersibility in the aqueous matrix and instability of bioactive compounds is challenging for food application. This study performed the physicochemical and chemical characterization of quinoa oil and evaluated its water dispersibility and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity after nanoencapsulation in porcine gelatin and combination with whey protein isolate by emulsification O/W technique. Thus, three formulations were obtained: 1) OG-containing quinoa oil and porcine gelatin in aqueous phase 2; 2) OWG1-containing quinoa oil, whey protein isolate, and porcine gelatin in aqueous phase 2; and 3) OWG2-containing quinoa oil and whey protein isolate in aqueous phase 1, and porcine gelatin in aqueous phase 2. The oil characterization showed that quinoa oil presented the predominance of linoleic acid (53.4%), and concentration of alpha and gamma-tocopherol, respectively, of 8.56 and 6.28 mg.100g-1. All formulations presented a smooth surface without depression or cracking, an average diameter between 165.77 and 529.70 nm. Fourier transform infrared spectroscopy indicated chemical interaction between the encapsulating agents and the oil in all formulations, being more intensified in OWG1 and OWG2. Based on this, these formulations showed higher dispersibility in aqueous solution [68% (3.48) and 71% (2.97)]. This resulted in higher antioxidant activity for OWG1 and OWG2, showing the amounts that reduces antioxidant activity by 50% equal to 5.30 (0.19) mg/mL and 5.54 (0.27) mg/mL, respectively, compared to quinoa oil [13.36 (0.28) mg/mL] (p < 0.05). Thus, quinoa oil nanoencapsulation proved to be an efficient alternative to enable water-dispersibility and enhance antioxidant activity, increasing its potential for application in the food industry.

Extraction of Carotenoids and Fat-Soluble Vitamins from Tetradesmus Obliquus Microalgae: An Optimized Approach by Using Supercritical CO2.

In recent years, great attention has been focused on rapid, selective, and environmentally friendly extraction methods to recover pigments and antioxidants from microalgae. Among these, supercritical fluid extraction (SFE) represents one of the most important alternatives to traditional extraction methods carried out with the use of organic solvents. In this study, the influence of parameters such as pressure, temperature, and the addition of a polar co-solvent in the SFE yields of carotenoids and fat-soluble vitamins from T. obliquus biomass were evaluated. The highest extraction of alpha-tocopherol, gamma-tocopherol, and retinol was achieved at a pressure of 30 MPa and a temperature of 40 °C. It was observed that overall, the extraction yield increased considerably when a preliminary step of sample pre-treatment, based on a matrix solid phase dispersion, was applied using diatomaceous earth as a dispersing agent. The use of ethanol as a co-solvent, under certain conditions of pressure and temperature, resulted in selectively increasing the yields of only some compounds. In particular, a remarkable selectivity was observed if the extraction was carried out in the presence of ethanol at 10 MPa and 40 °C: under these conditions, it was possible to isolate menaquinone-7, a homologous of vitamin K2, which, otherwise, cannot not recovered by using traditional extraction procedures.

Identification and evaluation of antioxidants in Japanese parsley.

Two cultivars of Japanese parsley were harvested in different seasons; their antioxidant capacities were evaluated by oxygen radical absorbance capacity (ORAC) methods, and the contents of hydrophilic and lipophilic antioxidants were compared. Japanese parsley possessed potent antioxidant capacities both in hydrophilic and lipophilic extracts when evaluated by ORAC methods. LC/MS/MS analyses revealed that chlorogenic acid and four kinds of quercetin glycosides were major antioxidants in the hydrophilic extract. Lutein was the main contributor to the antioxidant capacity of the lipophilic extract. Antioxidant capacities of the hydrophilic extracts of both cultivars tended to be higher in winter because of the increase in the contents of chlorogenic acid and quercetin glycosides. An obvious trend in the lipophilic antioxidant capacities or lutein contents was not observed irrespective of the cultivar.

Tocochromanols composition in kernels recovered from different apricot varieties: RP-HPLC/FLD and RP-UPLC-ESI/MS(n) study.

Composition of tocochromanols in kernels recovered from 16 different apricot varieties (Prunus armeniaca L.) was studied. Three tocopherol (T) homologues, namely α, γ and δ, were quantified in all tested samples by an RP-HPLC/FLD method. The γ-T was the main tocopherol homologue identified in apricot kernels and constituted approximately 93% of total detected tocopherols. The RP-UPLC-ESI/MS(n) method detected trace amounts of two tocotrienol homologues α and γ in the apricot kernels. The concentration of individual tocopherol homologues in kernels of different apricots varieties, expressed in mg/100 g dwb, was in the following range: 1.38-4.41 (α-T), 42.48-73.27 (γ-T) and 0.77-2.09 (δ-T). Moreover, the ratio between individual tocopherol homologues α:γ:δ was nearly constant in all varieties and amounted to approximately 2:39:1.

Sassarandainol: a new neolignan and anti-inflammatory constituents from the stem of Sassafras randaiense.

A new neolignan, (R)-( - )-sassarandainol (1), together with 10 known compounds (2-11), was isolated from the stem of Sassafras randaiense. The structures were determined by spectroscopic techniques. Among these isolates, γ-tocopherol (5), subamolide B (7) and ß-sitosterone (9) exhibited moderate iNOS inhibitory activity on nitrite production induced (%) value of 30.51, 28.68 and 16.96, respectively.

Buckwheat achenes antioxidant profile modulates Aspergillus flavus growth and aflatoxin production.

Buckwheat (Fagopyrum spp.) is a "pseudo-cereal" of great interest in the production of healthy foods since its flour, derived from achenes, is enriched with bioactive compounds and, due to the absence of gluten, may be used in composition of celiac diets. Amongst buckwheat species, F. tataricum achenes possess a larger amount of the antioxidant flavenol rutin than the common buckwheat F. esculentum. Ongoing climate change may favor plant susceptibility to the attack by pathogenic, often mycotoxigenic, fungi with consequent increase of mycotoxins in previously unexploited feeds and foodstuffs. In particular, Aspergillus flavus, under suitable environmental conditions such as those currently occurring in Italy, may produce aflatoxin B1 (AFB1), the most carcinogenic compound of fungal origin which is classified by IARC as Category 1. In this study, the viable achenes of two buckwheat species, F. tataricum (var. Golden) and F. esculentum (var. Aelita) were inoculated with an AFB1-producing A. flavus NRRL 3357 to analyze their relative performances against fungal invasion and toxin contamination. Notably, we sought the existence of a correlation between the amount of tocols/flavonols in the achenes of buckwheat, infected and non-infected with A. flavus, and to analyze the ability of the pathogen to grow and produce toxin during achene infection. Results suggest that achenes of F. tataricum, the best producer of antioxidant compounds in this study, are less susceptible to A. flavus infection and consequently, but not proportionally, to mycotoxin contamination compared with F. esculentum. Moreover, rutin-derived quercetin appears to be more efficient in inhibiting aflatoxin biosynthesis than the parent compound.

Automated measurement method for the determination of vitamin E in plasma lipoprotein classes.

In a subendothelial space of atherosclerotic arteries, apolipoprotein B-containing lipoproteins are accumulated and oxidized, and the oxidized lipoproteins promote macrophage foam cell formation. Therefore, the analysis of vitamin E, a major antioxidant in lipoproteins, is important for understanding atherosclerotic pathogenesis. A new method for the automated measurement of vitamin-E (γ- and α-tocopherols) in plasma HDL, LDL, and VLDL was established by using anion-exchange-chromatography for separation of lipoproteins, reverse-phase-chromatography for separation of γ- and α-tocopherols in each of lipoproteins, and fluorescent detection. The within-day assay and between-day assay coefficients of variation for lipoprotein tocopherol levels were 4.73-12.84% and 7.00-14.73%, respectively. The γ- and α-tocopherol/cholesterol ratios of VLDL were higher in healthy plasma than in plasma of untreated patients with dyslipidemia, but the ratios of LDL and HDL were not different. This new estimated method can provide the reliable data of lipoprotein vitamin-E and would be useful for the clinical settings.

Evaluation of vacuum microwave-assisted extraction technique for the extraction of antioxidants from plant samples.

In the present work, vacuum microwave-assisted extraction (VMAE) was to perform microwave-assisted extraction in vacuum. Two well-known antioxidants, vitamin C from guava and green pepper, and vitamin E (alpha-tocopherol and gamma-tocopherol) from soybean and tea leaves, which were easy to be oxidized, were chosen as representative target compounds for the evaluation of VMAE. The extraction yields of vitamin C, alpha-tocopherol and gamma-tocopherol in VMAE and those in MAE performed in atmosphere (air-MAE) were compared and the effects of extraction time, extraction temperature and sample matrix were studied. Moreover, the effects of the oxygen and subpressure invacuo were also discussed via performed MAE in N(2) atmosphere (N(2)-MAE). The extraction yields of vitamin C, alpha-tocopherol and gamma-tocopherol in VMAE were higher than that in air-MAE, 35% increments of vitamin C from green pepper, 22% increments of alpha-tocopherol and 47% increments of gamma-tocopherol from tea leaves were obtained, respectively. The comparable increased extraction yields of vitamin C, alpha-tocopherol and gamma-tocopherol in N(2)-MAE to that in air-MAE confirmed that oxygen in system was the crucial factor for the oxidation of vitamin C and vitamin E, VMAE was beneficial for the extraction of these oxygen-sensitive compounds. In addition, the subpressure invacuo in the VMAE system also showed positive affect on the extraction yields. On the basis of preventing oxidation and improving extraction efficiency of target compounds because of less oxygen and subpressure invacuo in the extraction system, VMAE has good potential for the extraction of oxygen-sensitive and thermosensitive compounds from plant samples.

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography.

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.

Comparison of extraction methods for quantifying vitamin E from animal tissues.

Four extraction methods: (1) solvent (SOL), (2) ultrasound assisted solvent (UA), (3) saponification and solvent (SP), and (4) saponification and ultrasound assisted solvent (SP-UA), were used in sample preparation for quantifying vitamin E (tocopherols) in chicken liver and plasma samples. The extraction yields of SOL, UA, SP, and SP-UA methods obtained by adding delta-tocopherol as internal reference were 95%, 104%, 65%, and 62% for liver and 98%, 103%, 97%, and 94% for plasma, respectively. The methods with saponification significantly affected the stabilities of tocopherols in liver samples. The measured values of alpha- and gamma-tocopherols using the solvent only extraction (SOL) method were much lower than that using any of the other extraction methods. This indicated that less of the tocopherols in those samples were in a form that could be extracted directly by solvent. The measured value of alpha-tocopherol in the liver sample using the ultrasound assisted solvent (UA) method was 1.5-2.5 times of that obtained from the saponification and solvent (SP) method. The differences in measured values of tocopherols in the plasma samples by using the two methods were not significant. However, the measured value of the saponification and ultrasound assisted solvent (SP-UA) method was lower than either the saponification and solvent (SP) or the ultrasound assisted solvent (UA) method. Also, the reproducibility of the ultrasound assisted solvent (UA) method was greater than any of the saponification methods. Compared with the traditional saponification method, the ultrasound assisted solvent method could effectively extract tocopherols from sample matrix without any chemical degradation reactions, especially for complex animal tissue such as liver.

Determination of antioxidant components in rice bran oil extracted by microwave-assisted method.

Rice bran oil was extracted by microwave-assisted extraction with isopropanol and hexane using a solvent-to-rice bran ratio of 3:1 (w/w). The experiments were done in triplicate at 40, 60, 80, 100, and 120 degrees C with a total extraction time of 15 min/sample. The oil components were separated by normal-phase HPLC and quantified with a fluorescence detector. The radical scavenging capability of the oil was tested with DPPH and was expressed as mumol Trolox Equivalent Antioxidant Activity. The increase in total vitamin E with temperature from 40 to 120 degrees C was 59.63% for isopropanol and 342.01% for hexane. Isopropanol was the best solvent for the extraction of gamma-tocopherol and gamma-tocotrienol as compared with hexane for both microwave-assisted and conventional solvent extraction. Isopropanol was better for oil yield extraction at high temperatures. Samples extracted with isopropanol at 120 degrees C had higher antioxidant activity. No differences in oil yield, total vitamin E, and antioxidant activity of oil was noticed between the two methods (microwave-assisted and solvent extractions), at 40 degrees C. No degradation of alpha-tocopherol was noticed during the process.

Reinvestigation of the effect of heat pretreatment of corn fiber and corn germ on the levels of extractable tocopherols and tocotrienols.

We previously reported that heat pretreatment of corn fiber (150 degrees C, 1 h) caused a tenfold increase in the levels of extractable gamma-tocopherol. The current study was a reinvestigation of the previous effect, using improved methods (HPLC with fluorescence detection, diode-array UV detection, and mass spectrometry) for tocol analysis. Heat pretreatment did not cause an increase in the levels of any of the tocopherols or tocotrienols in corn fiber oil, but lowered the levels of three of the tocols and had no effect on the levels of the other two tocols. Heat pretreatment of corn germ had a similar effect. UV and mass spectra indicated that the peak that we had identified as gamma-tocopherol in our previous report was probably a mixture of oxidation products of triacylglycerols. Thus, heat treatment of corn germ or other corn-oil containing fractions at high temperatures leads to decreases in gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol and to the production of triacylglycerol oxidation products.

Cyclodextrin-modified microemulsion electrokinetic chromatography for separation of alpha-, gamma-, delta-tocopherol and alpha-tocopherol acetate.

Different forms of tocopherols, together with tocotrienols, are collectively named as vitamin E, and each possesses different degree of medical, biological and physiochemical significance. The main difficulty of separating different forms of tocopherols lay in their highly structural similarities and hydrophobicities. Microemulsion electrokinetic chromatography (MEEKC), claimed to attain high peak efficiency with great solubilization power, has not previously been applied to the separation of tocopherols. The effects that various parameters, such as buffer system, type and concentration of cyclodextrins, temperature, and sample matrix, have on the separation of tocopherols by MEEKC have been investigated. By using a buffer mixture of 4% (w/w) sodium dodecyl sulfate (SDS), 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane, 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25mM, pH 2.5), and 25mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), the separation of alpha-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, as well as the antioxidant butylated hydroxytoluene (BHT) at -26kV, 25 degrees C was completed within 35min. The practical potential of the present approach has been further validated by the determination of tocopherols in a vitamin E preparation, with the result of 132.63 (RSD 1.25%), 176.51 (RSD 0.29%), and 64.32mg (RSD 3.34%) per 500mg capsule for alpha-, gamma- and delta-tocopherol, respectively.

Separation of delta-, gamma- and alpha-tocopherols by CEC.

In this study capillary electrochromatography (CEC) was used for the separation of three tocopherols (TOHs), namely delta-, gamma- and alpha-TOH and the antioxidant compound, butylated hydroxytoluene (BHT). The CEC experiments were carried out using an octadecylsilica (ODS) stationary phase packed, in our laboratory, in a fused-silica capillary (100 microm I.D., 365 microm O.D. x 33 cm of total length and 24.6 or 8.4 cm effective length). The mobile phase was composed by a mixture of methanol (MeOH) and acetonitrile (ACN), at different concentrations and 0.01% (w/v) of ammonium acetate. Retention time (t(R)), retention factor (k), resolution (R(s)) of the three TOHs were strongly influenced by the organic solvent composition of the run buffer and by the effective length of the capillary. Optimum experimental conditions were found even employing the short effective length of the capillary achieving the baseline separation of the studied analytes in a relatively short time (less than 5 min). The optimized method was applied to the qualitative analysis of vitamin E (alpha-TOH) present in a human serum extract.