Studies On The Functional Activities Of Tong-seed Meal And Eucommia Ulmoicles Oliv Extracts

In the last 20 years, active oxygen and free radicals have become the hot topicsof modern life science, and the appraisal and selection of natural resources with strongantioxidative activities have become the new trends of biology, medical and foodscience and technology. And with the passing epoch of antibiotics, the search ofeffective and harmless antibiotic substitute has become the basic point for thesustainable development of husbandry. Therefore, for the sake of the health of humanbeing and animals, the exploitation of antibiotic alternatives without remnant,pollution, toxic and side effects becomes increasingly important. Botanicals are thecritical resource of natural antioxidants and feed additives. China holds the title ofbotanic kingdom with various species, so it is obviously meaningful to research on thecertain uninvestigated or being little investigated herbals.Eucommia ulmoides Oliv, as an important Chinese herbal medicine, abound withiridoids such as aucubin, eucommiol, geniposide, geniposidic acid, phenols such aschlorogenic acid, lignans, coniferin, syringin, koaburaside, flavonoids and terpenes.Tong-seed meal extract extracted with 95% ethanol was reported abundant withpolyphenols, so both are important sources for the development of natural feedadditives and antioxidants. Though animal feeding and slaughter tests and oxidationkinetic characteristictest of pig concentrate, the substitution role of EUOE asantioxidants and antibiotics was investigated and the antioxidation and hypolipidemiceffects of the Tong-seed meal extract were appraised at whole organism level and celllevel in vitro and in vivo according to the high throughput screening. The activesubstance was extracted from the Tongmeal extract, and its main active componentswere further isolated and purified and their basic structures were identified. The mainexperimental results were as follows:(1) To investigate the effect of Eucommia ulmoicles Oliv Extract (EUOE) ongrowth performance, carcass characteristics, meat qualities and muscular amino acidcontents, a feeding trial was conducted with three hundred 42-d Durocx (LandracexYorkshire) crossbred piglets with average weight 9.96±0.05 kg. They were randomlyassigned to five treatment groups (six replicates per group, ten heads per replicate).The trial was spit into three phases: Phaseâ… (9.96-20kg BW); Phaseâ…¡(20-60kgBW) and Phaseâ…¢(60-90kg BW). The piglets were fed on the basal dietsupplemented with graded EUOE (0, 1500, 2000, 2500mg/kg) or flavomycin(12mg/kg for Phaseâ… and 8mg/kg for Phaseâ…¡andâ…¢) respectively, the diet supplemented with 1500 mg/kg EUOE showed significantly better results of ADDS(Phaseâ… ) (P＜0.05), ADG, F/G and ADDS (Phaseâ…¡) (P＜0.05), F/G (Phaseâ…¢) (P＜0.05)than that of the control, and there was a trend of increase on ADFI with the gradedlevel of EUOE supplementation (P=0.074) (Phaseâ…¢). As for the overall phase, ADGof 1500, 2500mg/kg EUOE and Flavomycin treatments were significantly differentfrom the control (P＜0.05), no significant difference existed on other indices but atrend is detected on ADFI (P=0.086). Compared with the control, TSC and SUNcontents of all the EUOE treated groups were significantly decreased during all thethree periods (P＜0.05). SG contents were increased during Phaseâ…¡,â…¢(P＜0.05).There was a trend that EUOE supplementation improved the carcass characteristicsand meat quaiities by decreasing the drip loss, skin thickness and increasing lion eyearea (0.05＜P＜0.10). TMAA contents of EUOE and Flavomycin treatments weresignificantly increased, while DMAA contents of Flavomycin was significantlylowered but that of 1500 mg/kg EUOE was significantly increased in pork muscle(P＜0.05). Furthermore, the examined DMAA and TMAA contents were decreasedwith the graded EUOE level (P＞0.05). These results indicated that EUOEsupplementation and Flavomycin had no significant difference on the growthperformance (P＞0.05), but better results of EUOE on carcass qualities and DMAAcontents were observed. The comparatively optimal dietary EUOE level was1500mg/kg. It suggested that EUOE could be an attractive antibiotic alternative foranimal nutrition, although further experiments are necessary to verify the results.(2) Antioxidant capacity comparison of four kinds of antioxidants includingEUOE showed that significant difference was found with the addition of antioxidants.The higher the addition level of feed fat, the higher the feed POV, and the higherantioxidant level was needed. All the antioxidants activities was significantlyincreased by the antioxidants whether or not the level of the fat level of the pigconcentrate but the activities of the botanical feed mainly exhibited the first 21st day,and no significant effect were detected after the 28th day. Based on the POV curves,EMQ(I) showed the best antioxidant capacity, while the EUOE activity was ratherlower. Different antioxidants and their addition levels differed the oxidation kineticsequations of the concentrate, as to EUOE, different EUOE levels and the fat levels ofpig concentrate mainly caused the Class One reaction of oxidation.(3) One monomer was isolated from 50% ethanol fraction of 95% ethanol extractof Tongmeal through NKA-9 resin. With application of HPLC and concerned physical and chemical characteristics, the structure of the monomer was identified by means ofUV, IR, MS, ¹HNMR and ¹³CNMR. It was named as (±)3,3ï½€-bisdemethylpinoresinol,which was first purified from Tong-seed meal.(4) High throughput screening was used to detect the anti-diabetes, anti-hyper-lipidenmia and anti-tumor capacities of the extracts. The results showed: 70%ethanol fraction at the levels of 50μl/ml and 100μl/ml had the abilities of inhibitoryrole on 28SC tumor cell and water fraction at the levels of 10μl/ml and 50μl/ml, and70% ethanol fraction at the level of 50μl/ml on HL-60 tumor cell. The monomer and50% ethanol fraction showed strong inhibitory effect on SMMC-7721 at the level of50μg/ml. 50% ethanol fraction had the anti-diabetes activity at the level of 50μg/mlwith the activation multiple to PPARa,PPARd,PPARγ, RXR receptor at 1.29, 3.88,7.06 and1.95 respectively.(5) After the experiments of the different ethanol fraction on antioxidantcapacities under different system in vitro, the total phenol contents differedsignificantly (P＜0.05) from 47.68 mg/g of 50% ethanol fraction to 34.51mg/g ofwater fraction. The scavenging capacity of DPPH·differed from one another at thehighest ability of 90.37% of 50% ethanol fraction. The deoxidizing capacity of thethree fractions of 30%, 50% and 70% ethanol fraction were significantly higher thanthat of the vitamin C (0.2 mg/ml) and BHT (0.2 mg/ml) at the level of more than 0.8mg/ml. And the inhibitory abilities of pyrogallol auto-oxidation of the three fractionsamounted to 96.67% with the equivalent ability of vitamin C at the same level of 0.20mg/ml (P＞0.05). As to the inhibitory capacity of linoleic acid peroxidation, at thelevel of 0.50 mg/ml, 50% ethanol fraction and vitamin C had the similar effects(P＞0.05). At the level of 0.80 mg/ml, the clear ability of 50% ethanol fraction tohydroxyl radicals amounted to 96.01%. As the increase of the concentration of thethree fractions, the hemolysis inhibitory percent of mice RBC induced by H₂O₂increased, and at the different concentrations, 50% ethanol showed the best effect.(6) Fifty Kunming SD mice were randomly arranged into 5 treatments (10 miceper treatment) to conduct the anti-hyperlipidenmia test based on the body weight. Onetreatment was fed on the common diet, while the other treatments were fed on high-fat feed. After the success of hyperlipidenmia model, three treatments fed on high-fatfeed were lavaged with 50% ethanol fraction at the levels of 1000, 500, 250 mg/kgBW respectively. The experimental resuies showed that 50% ethanol fraction couldeffectively decrease blood lipids level of SD mice with hyperlipemia, improve blood lipid metabolism, reduce the formation of atherosclerosis, effectively increaseantioxidative level of mice body, and improve antioxidase activities. The three levelsof 50% ethanol fraction significantly decreased TG, TC, LDL-C, ApoB, R-CHR,TC-HDL-C/HDL-C and LDL-C/HDL-C (P＜0.05), and significantly increased HDL-Cand Apo-AI level (P＜0.05), and significantly increased mice serum and liver TAC,SOD,GSH-Px activities,hydroxyl radicals scavenging capacities, decreased MDAcontents (P＜0.05). The three levels of 50% ethanol fraction could significantlydecrease the levels of blood viscosity, plasma viscosity, fibrinogen, hematocrit,indices of erythrocyte rigidity, aggregation (P＜0.05), and increase indexes oferythrocyte deformability (P＜0.05).