| In this study, the extraction method of Cordyceps militaris carotenoid was optimized, and studys that the effects of Cordyceps militaris carotenoid extracts (CMCE) on growth, immunity and oxidation resistance in mice and on the inhibition of oncocytes were carried out through animal experiments and cell culture experiments, including three parts (four experiments). The abstracts of there researches are as follows, espectively:Part one. By taken extraction efficiency of carotenoids from Cordyceps militaris as an index, six different extraction approaches were compared for selecting the best one, they were heat acid extraction technique, ultrasonic wave extraction technique, heat acid-ultrasonic wave extraction technique, microwave extraction technique, heat acid microwave extraction technique and acetone extraction technique. The results showed that heat acid-ultrasonic wave extraction technique was the better one for extract carotenoids in C.militaris. The optimum approach was adopted to conduct six single-factor experiments, including HCl concentration, time of soaking in HCl, heating time, ultrasonic power, ultrasonic extraction temperature and ultrasonic extraction time, then the L27(313) orthogonal test was designed to optimize the extraction conditions. Next, CMCE were identified with HPLC-MS technology. The results showed that, (1) the extraction efficiency could reach up to 1134.88 μg/g under these conditions, they were the 2.25 mol/L concentration of HCl,30 min of soaking time in HCl,400 W of ultrasonic power,6 min heating time,25 ℃ ultrasonic extraction temperature and 25 min ultrasonic extraction time; (2) These conditions ranked as follows in turn:ultrasonic power, time of soaking in HCl, HCl concentration, heating time, ultrasonic extraction temperature, ultrasonic extraction time. Part two. The effects of different concentration and different kinds of CMCE on growth, immunity and oxidation resistance in mice, respectively. Including two experiments, as follow:Experiment one. The effects of different concentrations of CMCE on growth, immunity and oxidation resistance in mice. One-hundred-and-twenty ICR mice were randomly divided into four groups. Each group was fed a different concentration of CMCE solution (0.2 ml per animal per day), i.e.,0 (Group C),2 (Group L), 4 (Group M) and 8 mgml-1 (Group H), respectively. The results indicated that, (1) halfway through the experiment (at 14 days), the ADG values of Groups L and M were very significantly higher than those of the control (C) and H (P<0.01) groups and the FIG of Group L was significantly lower than that of Group C (P<0.05). After 28 days, the ADFI were significantly higher (P<0.05) and ADG were very significantly higher (P<0.01) than Group C and in Group L and M. Low and Middle concentration of CMCE could help to increase the digestibility of DM, the CHO and TG inserum in mice, increase the V/C (Villus height/Crypt depth) values, (2) and could increase TNF-α mRNA relative expression values (P<0.01); However, High concentration of CMCE would increase the content of IgG in serum (P<0.01), would increase IL-2 mRNA relative expression values (P<0.01) in spleen and decrease V/C values in three part of intestine; (3) The activity of serum CAT and GSH-Px in Group L was very significantly higher (P<0.01) than other Groups; The activity of serum MDA in Group M was significantly higher (P<0.05) than Group H. The activity of liver SOD in Group L was significantly higher (P<0.05) than Group C; The activity of liver CAT showed dose-dependent effect and the activity of liver CAT in Group H was significantly higher (P<0.05) than Group C.Experiment two. The effects of carotenoids on growth, immunity and oxidation resistance in mice. Forty-eight ICR mice were randomly divided into four groups. Each group was fed a different carotenoids solution (0.2 ml per animal per day), i.e., Soybean oil solution (Group A), P-carotenoid (Group B), lutein (Group C) and CMCE (Group D), respectively. The results indicated that, (1) after 28 days, the ADFI values of Groups D was significantly higher than that of control group (P<0.05) and there was no significantly difference among four groups about the vales of ADG and F/G (P>0.05); GLU and CHO of Group D were very significantly (P<0.01) and significantly (P<0.05) higher than Group A respectively; The content of TG of Group B and C were significantly (P<0.05) and very significantly (P<0.01) higher than Group D respectively; (2) The content of IgG in Group B was significantly higher than Group D (P<0.05), WBC and Lymph of Group D were both very significantly higher than Group A (P<0.01) and the Mon and Mon% of Group D were also significantly higher than Group A (P<0.05); in Group B, the relative expression of IL-2 mRNA was significantly higher (P<0.05) than in Group A; the relative expression of IL-4 and TNF-a in Group D were significantly higher than in Group C and Group A (P<0.05); (3) The activity of serum SOD in Group B was ignificantly higher than in Group C and D and the activity of serum CAT and GSH-Px of Group B were significantly (P<0.05) and very significantly (P<0.01) higher than Group D; The activity of serum GSH-Px in Group C was very significantly (P<0.01) higher than other three groups and Group C and D were very significantly (P<0.01) lower than Group B and A as regards the content of serum MDA. In liver, there were no significant difference among four groups as regards SOD, CAT and GPX1 mRNA relative expression (P>0.05).Part three. The preliminary purification CMCE was used to illustrate the growth inhibition of gastric cancer (MGC80-3) and hepatoma cell (HepG2) in vitro. The concentration of carotenoids in CMCE was improved the 12 times after preliminary silica gel column chromatography purification. Different concentration of CMCE solution which were 60,80,100,120 and 140μg/mL, respectivly, were prepared. Cis-platinum was for positive control group and culture medium without CMCE was for negative control group. The results indicated that, (1) after culturing for 24 hours, the effect of CMCE on the growth inhibition of MGC80-3 showed dose-dependent. After 48 and 72 hours,80 and 100μg/mL CMCE had the major effect on growth inhibition of MGC80-3. (2) To HepG2, after culturing for 24 hours, the effect of CMCE on the growth inhibition of HepG2 showed dose-dependent; After 72 hours,120μg/mL CMCE had the major effect on growth inhibition of HepG2. |
