Dietary butylated hydroxytoluene improves lipid metabolism, antioxidant and anti-apoptotic response of largemouth bass (Micropterus salmoides).

A 10-week growth trail was conducted to investigate the efficacy and tolerance of dietary butylated hydroxytoluene (BHT) by evaluating inflammation, apoptosis and hepatic disease related to oxidative stress in largemouth bass (Micropterus salmoides). Four experimental diets were prepared with BHT supplement levels of 0 (B0), 150 (B150), 300 (B300) and 1500 (B1500) mg/kg, in which B150 was at the maximum recommended level established by European Union Regulation, and the B300 and B1500 levels were 2 and 10-fold of B150, respectively. Each diet was fed to 6 replicates with 30 largemouth bass (initial body weight, IBW = 6.20 ± 0.01 g) in each tank. The BHT inclusion level did not affect the specific growth rate, but fish in the B150 group showed the lowest feed conversion rate (P < 0.05). BHT inclusion significantly decreased the levels of plasma TC, TG, LDL, ALT and AKP, and increased the (HDL-C)/TC ratio (P < 0.05). Plasma MDA was significantly decreased in the B150 group and GSH-Px was extremely enhanced in each BHT inclusion group (P < 0.05). Hepatic T-AOC was significantly enhanced and O2- was significantly decreased in each BHT inclusion group compared to the B0 group (P < 0.05), as well as hepatic MDA was significantly decreased in B1500 group (P < 0.05). Dietary BHT inclusion down-regulated the hepatic mRNA levels of inflammation, apoptosis and fibrosis related genes, including TNFα, TGF-ß1, α-SMA, IL8, IL11ß and caspase-9. Moreover, BHT could improve hepatic lipid metabolism via up-regulating the mRNA levels of APOA1, CYP7A1, CYP8B1, and down-regulating the mRNA levels of PPAR-γ and APOB. Histological examination of the liver morphology with H&E and Sirius Red staining showed that BHT inclusion decreased necrotic degenerative changes and collagen deposition in largemouth bass. An immunofluorescence examination revealed significantly decreased cleaved caspase-3 signals in the BHT groups. In conclusion, the results demonstrated that ROS induces hepatic cell apoptosis and fibrosis via the intrinsic pathway of apoptosis by activating caspase-9 in the mitochondria and then initiates apoptosis by activating caspase-3. Consuming 2.32-23.80 mg/kg·bw/d (150-1500 mg/kg in diet) of BHT effectively improved the plasma and hepatic lipid metabolism, antioxidant response as well as reduced ROS production, protecting hepatic cells from injury. It is implied that even a 10-fold increase of the maximum level of BHT (150 mg/kg) is safe for the largemouth bass.

In ovo feeding of creatine pyruvate alters energy reserves, satellite cell mitotic activity and myogenic gene expression of breast muscle in embryos and neonatal broilers.

We investigated the effects of in ovo feeding (IOF) of creatine pyruvate (CrPyr) on energy reserves, satellite cell mitotic activity (SCMA) and myogenic gene expression in breast muscle of embryos and neonatal broilers. A total of 960 eggs were randomly allocated into three treatments: 1) non-injected control group, 2) saline group injected with 0.6 mL of physiological saline (0.75%), and 3) CrPyr group injected with 0.6 mL of physiological saline (0.75%) containing 12 mg CrPyr/egg at 17.5 d of incubation. After hatching, a total of 120 male chicks were randomly assigned to each treatment group, with eight replicate sets per group. Selected chicks had body BW close to the average of their pooled group. Our results showed that the total and relative breast muscle weights of broilers subjected to CrPyr treatment were higher than those in the control and saline groups on 19 d of incubation (19 E), the day of hatch, 3 and 7 d post-hatch (P < 0.05). The myofiber diameter and cross-sectional area of individuals in the CrPyr group were higher than those in other treatments on 3 and 7 d post-hatch (P < 0.05). Moreover, IOF of CrPyr increased (P < 0.05) creatine concentrations on 19 E, the day of hatch and 3 d post-hatch, the same treatment increased phosphocreatine concentrations on 19 E. Broilers in the CrPyr group showed higher expression of myogenic differentiation 1 (MyoD) (P < 0.05), myogenin and paired box 7 (Pax7), as well as higher index of SCMA on 3 d post-hatch. However, myostatin mRNA expression in CrPyr-treated broilers was down-regulated on 3 d post-hatch (P < 0.05). These results indicated that IOF of CrPyr increased energy reserves of embryos and SCMA of broilers on 3 d post-hatch, which led to enhanced muscle growth in the late embryos and neonatal broilers. Additionally, IOF of CrPyr increased the activity of satellite cells possibly through up-regulating MyoD, myogenin, and Pax7 mRNA expression and down-regulating myostatin mRNA expression.

Potential of a novel polysaccharide preparation (GLPP) from Anhui-grown Ganoderma lucidum in tumor treatment and immunostimulation.

Growing evidence indicates the potential of developing novel polysaccharide-based adjuvant for tumor therapy from edible mushrooms, including Ganoderma lucidum. In the present study, a novel polysaccharide preparation (GLPP) was isolated from the fruiting body of G. lucidum grown in Anhui, China, and characterized for its physicochemical properties. GLPP had an average molecular weight of 6600 and a specific optical rotation of +25.6 degrees , contained 10.6% protein, and had a molar ratio of 0.9:15:1 for mannose, glucose, and galactose, respectively. GLPP was also investigated and compared with PSP (polysaccharopeptide preparation), a commercial antitumor and immunostimulating agent, for its antitumor and immunostimulation capacity, and potential in reducing the toxic effects induced by cyclophosphamide (Cy) treatment and Cobalt-60 ((60)Co) radiation in mice. GLPP at levels of 100 and 300 mg/kg body weight (BW)/d significantly inhibited the growth of inoculated S(180), Heps, and EAC tumor cells in mice. GLPP at a dose of 300 mg/kg BW/d showed stronger growth inhibition against all 3 tested tumor cells than PSP at 1 g/kg BW/d. GLPP also dose-dependently increased phagocytic index, phagocytic coefficient, and 50% hemolysin value in the EAC tumor-bearing mice, indicating its potential immunostimulating property. In addition, GLPP at 300 mg/kg BW/d was comparable to PSP at 1000 mg/kg BW/d in preventing the decrease of thymus index, spleen index, white blood cells, and bone marrow karyote numbers induced by Cy treatment and (60)Co radiation. These data demonstrated the potential utilization of GLPP as an adjuvant to conventional treatments of cancers and its use for cancer prevention.

Anti-diarrheal effect of water extract of Evodiae fructus in mice.

Our previous study showed that Evodiae fructus (the dried, unripe fruit of Evodia rutaecarpa) has an inhibitory effect on the intestinal transit (anti-transit effect) in mice. In the present study, a water extract of Evodiae fructus was used to examine its effect on castor oil-induced diarrhea and to compare with its anti-transit effect in mice. The results indicated that Evodiae fructus had both anti-transit and anti-diarrheal effects with comparable ID(50) (the dose for 50% inhibition) values of 54+/-7 and 76+/-17 mg/kg. The time-courses of Evodiae fructus pretreatment for both anti-transit and anti-diarrheal effects were very similar. Because no significant influences of both nitric oxide (NO) precursor L-arginine (600 mg/kg, i.p.) and NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (25 mg/kg, i.p.) pretreatment, the NO system was not involved in both the anti-transit and anti-diarrheal effects of Evodiae fructus. Like Evodiae fructus, a muscarinic acetylcholine receptor antagonist atropine inhibited castor oil-induced increase in fecal weight and loss of body weight. However, the potencies or time-courses of atropine pretreatment for both anti-transit and anti-diarrheal effects were different. Furthermore, the anti-diarrheal effect of atropine was independent of its anti-transit effect at the lower dose (0.5 mg/kg, i.p.). Therefore, the action of Evodiae fructus appeared to be something different from atropine, suggesting that an action other than the anti-muscarinic action, as previously proposed for Evodiae fructus, may be involved.

Central inhibitory effects of water extract of Acori graminei rhizoma in mice.

The present study evaluated in mice the central inhibitory effects of a water extract of shichangpu (Acori graminei rhizoma (AGR), the dry rhizome of Acorus gramineus Soland. (Araceae)). AGR (0.5-5.0 g/kg) dose-dependently decreased the locomotor activity and increased the pentobarbital-induced sleeping time, but had no significant effect on the treadmill performance. AGR also dose-dependently inhibited the intensity of apomorphine-induced stereotypic behavior. At the highest dose (5.0 g/kg), AGR had a weak anticonvulsant effect on the pentylenetetrazol-induced seizures. Receptor binding assays showed that AGR competed with [3H]SCH-23390 and [3H]YM-09151-2 for specific binding to striatal dopamine D1 and D2 receptors with Ki values of 5.6 and 4.2 mg/ml, respectively. AGR also competed with [3H]muscimol for specific binding to the gamma-aminobutyric acid (GABA) binding site of cortex GABA(A) receptors with a Ki value of 0.31 mg/ml. It also increased the specific binding of [3H]flunitrazepam to the benzodiazepine binding site of the GABA(A) receptors, suggesting a GABA agonist-like action. These results suggested that the central inhibitory effects of AGR were probably effected through an action on the central dopamine receptors and GABA(A) receptors. The principle of AGR acting at these ligand binding sites was not alpha-asarone, one of the important principles of AGR, since that alpha-asarone (10(-6)-10(-4) M) had no significant interactions with these binding sites.

6-Methoxy-N-methyl-1,2,3,4-tetrahydro-beta-carboline from Evodiae Fructus.

A receptor binding assay directed separation has led to the identification of a minor alkaloid 6-methoxy- N-methyl-1,2, 3,4-tetrahydro-beta-carboline (1) from the Chinese herbal drug Evodiae Fructus [EF, the dried, unripe fruit of Evodia rutaecarpa Look. f. et Thomas (Rutaceae)]. The structure of compound 1 was elucidated by means of spectroscopic methods and a comparison with synthetic materials. Compound 1 interacted with 5-HT (1A) and 5-HT (2) receptors with a moderate K (i) value of 78 and 1.2 microM, respectively. Compound 1 is found in EF and the genus Evodiae for the first time.

Evaluation with receptor binding assay on the water extracts of ten CNS-active Chinese herbal drugs.

In the present study, we established receptor binding assays to evaluate the water extracts of ten central nervous system (CNS)-active Chinese herbal drugs. These ten herbal drugs are Chaihu (Radix Bupleuri), Chuanxiong (Rhizoma Chuanxiong), Danggui (Radix Angelicae sinensis), Danshen (Radix Salviae miltiorrhizae), Duhuo (Radix Angelicae pubescentis), Hangqin (Radix Scutellariae), Qinjiao (Radix Gentianae macrophyllae), Shengma (Rhizoma Cimicifugae), Suanzaoren (Semen Zizphi spinose), and Yangjihua (Flos Daturae). The results indicated that these water extracts contained the principles acting on the dopamine (D1 & D2), muscarinice acetylcholine (M1), or 5-HT (5-HT1A & 5-HT2) receptors, or the benzodiazepine and the gamma-amino-n-butyric acid (GABA) binding sites of GABAA receptors as determined by receptor binding assays. The receptors or binding sites which predominantly acted by each water extract are listed as follows: Chaihu: D2, 5-HT1A, GABA; Chuanxiong: GABA, 5-HT1A; Danggui: GABA, 5-HT1A; Danshen: BDZ; Duhuo: GABA, 5-HT1A, D2, D1; Hangqin: BDZ, D1, 5-HT1A; Qinjiao: GABA, BDZ, 5-HT1A, D2; Shengma: 5-HT1A; Suanzaoren: 5-HT1A, 5-HT2, GABA; Yangjihua: M1, 5-HT1A, 5-HT2. These results provided evidence to explain the CNS effects of these herbal drugs at the receptor level. Furthermore, these results provided information to direct the isolation and purification of receptor-interactive compounds from these herbal drugs.

Effect of the crude extract of Evodiae Fructus on the intestinal transit in mice.

One of the uses of Evodiae Fructus (EF, the dried, unripe fruit of Evodia rutaecarpa) in Chinese medicine is recommended in diarrhea, but its underlying mechanism has not yet been studied. The present study examined the effect of an aqueous extract of EF on the intestinal transit in mice by the charcoal meal method. Intraperitoneal administration (i.p.) of EF (1.9-30 g/kg) significantly inhibited the intestinal transit in a dose- and time-dependent manner. This inhibitory effect of EF was not attenuated by the i.p. pretreatment with an alpha 2-, alpha 1-, or beta-adrenoceptor antagonist, i.e. yohimbine (10 mg/kg), prazosin (2 mg/kg), or propranolol (6 mg/kg), respectively. In the isolated mouse duodenum, jejunum, and ileum preparations, EF (10-50 mg/ml) concentration-dependently abolished 10 microM carbachol-induced contraction with an IC50 of 9.9, 11.7, and 16.3 mg/ml, respectively. This inhibitory effect was not competitive. Receptor binding assay showed that EF (1-50 mg/ml) significantly competed with [3H]-N-methylscopolamine for specific binding to muscarinic receptors on the duodenum, jejunum, and ileum membrane preparations with a Ki value of 7.1, 8.4, and 14.4 mg/ml, respectively. Therefore, the above results suggested that the inhibitory effect of EF on intestinal transit was probably via an action directly on the muscarinic receptors but not on the alpha 2, alpha 1-, and beta-adrenoceptors in the small intestine.