The preventive effect of ß-carotene on denervation-induced soleus muscle atrophy in mice.

Muscle atrophy increases the production of reactive oxygen species and the expression of atrophy-related genes, which are involved in the ubiquitin-proteasome system. In the present study, we investigated the effects of ß-carotene on oxidative stress (100 µM-H2O2)-induced muscle atrophy in murine C2C12 myotubes. ß-Carotene (10 µM) restored the H2O2-induced decreased levels of myosin heavy chain and tropomyosin (P< 0·05, n 3) and decreased the H2O2-induced increased levels of ubiquitin conjugates. ß-Carotene reduced the H2O2-induced increased expression levels of E3 ubiquitin ligases (Atrogin-1 and MuRF1) and deubiquitinating enzymes (USP14 and USP19) (P< 0·05, n 3) and attenuated the H2O2-induced nuclear localisation of FOXO3a. Furthermore, we determined the effects of ß-carotene on denervation-induced muscle atrophy. Male ddY mice (8 weeks old, n 30) were divided into two groups and orally pre-administered micelle with or without ß-carotene (0·5 mg once daily) for 2 weeks, followed by denervation in the right hindlimb. ß-Carotene was further administered once daily until the end of the experiment. At day 3 after denervation, the ratio of soleus muscle mass in the denervated leg to that in the sham leg was significantly higher in ß-carotene-administered mice than in control vehicle-administered ones (P< 0·05, n 5). In the denervated soleus muscle, ß-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P< 0·05, n 5) and the levels of ubiquitin conjugates. These results indicate that ß-carotene attenuates soleus muscle loss, perhaps by repressing the expressions of Atrogin-1, MuRF1, USP14 and USP19, at the early stage of soleus muscle atrophy.

Metabolism and absorption of auraptene (7-geranyloxylcoumarin) in male SD rats: comparison with 7-ethoxycoumarin.

We previously reported that auraptene (7-geranyloxycoumarin; AUR), a coumarin that occurs widely in citrus fruit, has been shown to be a promising cancer-preventive agent in several rodent models. However, its bioavailability and metabolism have not been investigated. In this study, we compared the metabolism characteristics of AUR with those of 7-ethoxycoumarin (ETC) in male Sprague Dawley rats. Each (500 micromol/kg body weight) was given separately by a single gastric intubation procedure, and digestive tract, liver, and kidney were removed at 1, 4, and 24 h after administration. The localization profiles of AUR and ETC in the gastrointestinal tract were similar. However, AUR, in contrast to ETC, showed significant localization in the liver from 1 to 4 h. Treatments of serum and urinary samples with glucuronidase/sulfatase led to the detection of significant amounts of umbelliferone (7-hydroxycoumarin; UMB), and serum and urinary concentrations of UMB following ETC administration were significantly higher than with AUR administration. Our results suggest that AUR, which bears a geranyloxyl side chain, has a longer life span than ETC, and this property may be associated with its previously reported chemopreventive and xenobiotics metabolizing activities.

Measurement of individual differences in intake of green and yellow vegetables and carotenoids in young unmarried subjects.

In this study, a dietary survey for 3 weekdays of young unmarried subjects [workers and students, 159 males and 160 females, ages 18-19y (17.2%), 20-24 y (56.2%), 25-29 y (18.6%), 30-34 y (6.7%) and 35 y- (1.3%)] was performed. We evaluated the intake of green and yellow vegetables in this survey and determined the carotenoids (beta-carotene, alpha-carotene, lycopene, beta-cryptoxanthin, and lutein+zeaxanthin) in 15 kinds of green and yellow vegetables frequently consumed. The carotenoid intake of each subject was calculated from the intake of these vegetables and the amount of carotenoid. Moreover, we studied the intake of protein, fat, and dietary fiber, and investigated its relationship to the intake of vegetables. The mean green and yellow vegetable intake/d in all subjects was 60.5 +/- 58.7 g, much lower than the recommended level [120 g/d, (Health Japan 21 by Japanese Ministry of Health, Labour and Welfare)]. The intake of green and yellow vegetables was greater in females than males, and in workers than students. In all subjects, the mean total carotenoid intake/d was 2852.8 +/- 2354.3 microg. In the total intake of carotenoids, there was no difference between males and females; however, the intake was greater in workers than in students. The intake of beta-carotene and a-carotene was greater in males than females. However, the intake of beta-cryptoxanthin, lycopene, and lutein+zeaxanthin was greater in females than males. The group with the low green and yellow vegetable intake had not only a low carotenoid intake, but also a low intake of protein, fat, and dietary fiber. Therefore, it was suggested that carotenoid absorption may be affected by a low intake of protein, fat, and dietary fiber.

Effects of administration of beta-carotene, ascorbic acid, persimmons, and pods on antioxidative ability in UV-irradiated ODS rats.

To evaluate the effects of supplementing diets with carotenoid and ascorbic acid (AsA) on the antioxidative ability of Osteogenic Disorder-Shionogi (ODS) rats, we added synthetic beta-carotene (betaC), AsA, and powders of persimmon (Ka) and pods (Po) containing betaC and AsA to the diet and obtained the following results. The urinary 8-hydroxydeoxyguanosine (8-OHdG) concentration was low in the -betaC.AsA and +AsA groups but high in the +betaC.AsA, +Ka, and +Po groups. The thiobarbituric acid-reactive substances (TBARS) in both the liver and skin were higher in the -betaC.AsA group than in the +betaC.AsA group and were low in the +Ka and +Po groups. As antioxidant enzymes, glutathione peroxidase (GSH-Px) activity was high in the +betaC.AsA group, low in the -beta3C.AsA group in both the skin and liver, and also high in the + Ka and +Po group in the liver. Superoxide dismutase (SOD) activity was high in the -betaC.AsA group and low in the +betaC.AsA and +Ka groups in both the skin and liver. Catalase (CAT) activity in the liver was low in the -betaC.AsA, +AsA, and +betaC groups and high in the +betaC.AsA and +Po groups. These results confirmed that the administration of betaC, AsA, and persimmons and pods increases antioxidative ability in the skin and liver of ultraviolet-b(UV-B)-irradiated ODS rats.

Effects of dietary protein, fat and beta-carotene levels on beta-carotene absorption in rats.

It is important that the factors influencing functional carotenoid absorption be made clear. The effect of rat diets containing different combinations of dietary protein, fat, and beta-carotene levels on beta-carotene absorption was evaluated. In the mid-level beta-carotene diet groups (1.8 mg beta-carotene/100 g diet), the retinol content in the liver and the ratio of the liver retinol content to the beta-carotene intake (namely, retinol accumulation rate) were both greater in the mid-level dietary protein groups (20 g protein/100 g diet) than in lower dietary protein groups (5 g protein/100 g diet), but were not affected by dietary fat level (10 g or 2 g fat/100 g diet). The liver beta-carotene content and its accumulation rate were lower in the mid-level dietary fat groups (10 g fat/100 g diet). beta-carotene 15,15'-dioxygenase (CDO) activity in the small intestine increased in the mid-level dietary protein groups, which resulted in an increase in the content of liver retinol converted from dietary beta-carotene. With respect to the mid-level dietary fat groups, the fecal beta-carotene excretion rate (%) increased. The low beta-carotene diet groups (0.006 mg beta-carotene/100 g diet) showed a higher serum retinol accumulation rate, CDO activity, and liver beta-carotene accumulation rate than the mid-level beta-carotene diet groups. These results suggest the marked effect of protein, fat, and beta-carotene level in diets on the absorption and metabolism of beta-carotene.

Effect of carotenoids and ascorbic acid of Japanese persimmons on cellular lipid peroxidation in HepG2 cells.

In this experiment, we examine the functional property of carotenoids; beta-cryptoxanthin (Cry), zeaxanthin (Zea), beta-carotene (Car)) and ascorbic acid (AsA). The accumulation amounts of Cry, Zea and Car in HepG2 cells cultured in the high concentration medium were larger than that in a low concentration. Further those accumulation amounts in long incubation time within 24 hours were greater than that in a shorter time. When the added carotenoid concentration, with or without hydrogen peroxide, increased from 0 to 5 microM in the culture medium, the thiobarbituric acid reaction substance (TBARS) values in the HepG2 cells decreased significantly (p < 0.05). The decrease of TBARS values shows the antioxidative property of the carotenoids. When AsA and Tocopherol(Toc) were added to the medium from 0 to 20 microM, the TBARS values, with or without hydrogen peroxide, decreased significantly with increasing concentrations of AsA and Toc respectively (p < 0.05). The decreased amount of TBARS in 5 microM Cry compared with control(0 microM) was the largest among 6 antioxidants (Cry, Car, Zea, Retinol(Ret), AsA, Toc) used in this experiment.

Improved simultaneous determination method of beta-carotene and retinol with saponification in human serum and rat liver.

Among the many simultaneous determination methods for carotenoid and retinoid, there are only a few reports including the saponification process. However, the yields of beta-carotene and retinol were higher when using this process. In this study, the analytical conditions, including saponification, were investigated. The extraction solvent was n-hexane and the sample solvent was HPLC mobile phase in the beta-carotene and retinol analysis. BHT as an antioxidant was added at concentrations of 0.125 and 0.025%, respectively, to ethanol and n-hexane phase in the extraction process for serum. The recovery rates were 99.7, 93.7 and 98.3% for beta-carotene, retinol and retinyl palmitate in serum, respectively, and 107.1, 92.8 and 98.8% for beta-carotene, retinol and retinyl palmitate in liver, respectively. The within-day coefficients of variation (C.V.) were 6.0% for serum and 4.7% for liver in the case of beta-carotene, 7.1% for serum, and 5.1% for liver in the case of retinol. The between-day coefficients of variation were 2.7% for serum and 2.7% for liver in the case of beta-carotene, and for retinol, 6.4% for serum and 2.7% for liver.

Characteristic rat tissue accumulation of nobiletin, a chemopreventive polymethoxyflavonoid, in comparison with luteolin.

Nobiletin (NOB), a polymethoxyflavonoid, is an effective anti-inflammatory and chemopreventive phytochemical found in citrus fruits. We compared the absorption and metabolism characteristics of NOB with those of luteolin (LT) in male SD rats. Each flavonoid (67.1 micromol/kg of body weight) was given separately by gastric intubation, and then concentrations were measured at 1, 4, and 24 hours after administration. In the digestive organs, NOB showed a notable tendency for localizing into the mucous membrane and muscularis from 1 to 4 hours, in contrast to LT, though both NOB and LT were completely excreted within 24 hours. Further, significant amounts of NOB were detected in the whole liver and kidney specimens, whereas LT accumulation was slight. Although serum concentrations of NOB from 1 to 4 hours were comparable to those of LT, urinary concentrations of LT were significantly higher from 4 to 24 hours. Following glucuronidase/sulfatase treatments of urinary materials, we detected 3 types of mono-demethylated NOB, including 3'-demethyl-NOB, and two di-demethylated types, as well as 3'-demethyl-NOB alone in serum samples using liquid chromatography-mass spectral analysis. Our results suggest that the metabolic properties of polymethoxyflavonoids are distinct from those of other general flavonoids, because of their wide distribution and accumulation in tissue.