Studies On Preparation And Bioactivities Of Inositol Trisphosphate

Bioactive inositol triphosphate(InsP3) was prepared from phytic acid by enzymatic or nonenzymatic ways. The enzymatic characteristics of different phytases and the hydrolysis conditions of phytic acid were investigated. Isolation and structural characterization of InsP3 were studied. The bioactivities of InsP3 were also investigated, including the antioxidant activities in vitro and in vivo, inhibition effect on S180 tumor growth and influence on immune ability in vivo, inhibition effect on human hepatocarcinoma cell line SMMC-7721, reduction effect on the level of blood glucose in diabetic mice induced by Alloxan. The main results are as follows:1 The enzymatic characteristics of wheat bran phytase and Aspergillusterrus phytasePhytase activity was expressed as the release of inorganic phosphate from phytic acid which was determined by Mo-Sb anti-spectrophotometry. Results showed that the optimum temperature, pH and concentration of substrate of phytate for Aspergillus terrus phytase to keep its activity were 50℃ 5.10 and 5mmol/L, respectively. Ca~2+ and Mg~2+ could stimulate its activity, but inorganic phosphate ion could inhibit its activity,with 27% phytase activity being repressed when the concentration of inorganic phosphate ion was about 10mmol/L. The Km value of Aspergillus terrus phytase for the phytate was 0.0528mmol/L.The phytase activity was about 1.48*10~5U/g under the optimum conditions.The optimum temperature, pH and concentration of substrate for wheat bran phytase activity were 37℃ 5.15 and 2.5mmol/L, respectively. Ca~2+ and Mg~2+ could stimulate its activity, but inorganic phosphate ion could inhibit its activity,with 24% phytase activity being repressed when the concentration of inorganic phosphate ion was about 10mmol/L. The Km value for the wheat bran phytase was 0.266mmol/L.The phytase activity was about 1.8U/g wheat bran under the optimum conditions. The optimum abstraction conditions for wheat bran phytase were as follows: the weight ratio between wheat bran and water was 1:6, temperature was 5℃ and abstraction time was about 2h. The wheat bran phytase should be stored at low temperature, when being treated at 37℃ for 16h, its activity was about 83.2% as that of phytase being newly dialyzed.2 Preparation and isolation of InsP3When the hydrolysis degree of phytic acid is about 50% by wheat phytase under its optimum conditions, the hydrolyzed solution was pumped into a column containing 717 strong anion-exchange resin, then delivered a linear gradient of 0.05-0.7M HC1. Thesamples of each tube were digested and the phosphate was determined. The contents of tubes corresponding to the same individual inositol phosphate were pooled together, and neutralized with NaOH , then precipitated by Ca2+.The calcium salt was then turned into InsP3 by 732 strong cation-exchange resin. The washed compound was neutralized with sodium hydroxide, rotary evaporated under vaccum, lyophilized. The InsP3 product prepared by this way contained no other low inositol phosphate by HPLC. The yield of InsP3 was about 28.4%. The InsP3 prepared could divided into two fractions of InsP3 by further isolization. The yield of InsP3 was about 26% by Aspergillus terrus phytase when the hydrolysis degree of phytic acid was about 50% and treated by the same way. Two fractions of InsP3 were also gotten by further isolization through 717 strong anion-exchange resin. The yield of InsP3 prepared by microwave radiation was about 18.4% when the hydrolysis degree of phytic acid was about 50% and treated by the same way above.The InsP3 prepared by wheat bran phytase, Aspergillus terrus phytase and microwave radiation were called InsP3A> InsP3B and InsP3C, respectivelyo3 Structural characterization of InsP3*HNMR Spectroscopy indicated that InsP3 prepared by wheat bran phytase was mainly Ins(l,2,3)P3 while those prepared by Aspergillus terrus phytase was mainly Ins(l,2,6)P3. The resonances were analyzed for the first by just one dimensional *HNMR.Electrospray (ESI) mass spectrometric method for the structural characterization of InsP3 was used. At pH 4.0, several [M + H]+ ions with different sodium atoms for Ins(l,2,6)P3 and Ins(l,2,3)P3 appeared. Ins(l,2,6)P3 and Ins(l,2,3)P3 yielded distinguishable spectra. Fragmentation pathways are dominated by the consecutive losses ofH2O, PO , HPO3 , HPO4 > RiPC^and CO. The mass spectroscopy were analyzed in details for the first time.IR Spectroscopy of InsP3 indicated that there were O-H and C-O-P structure in them.4 The antioxidant activities of InsP3 in vitro and in vivoInsP3 had lower reductive activity in vitro , but they can scavenge hydroxyl radicals in vitro and in the serum of mice. The sequence of capacity for scavenging hydroxyl radicals was as follows: InsP3A> InsP3 C>InsP3 B. At high concentration(0.4mg/mL),the scavenge rate of hydroxyl radical for InsP3 A, InsP3 B and InsP3 C were 91.54%, 90.78%, 91.73%,respectively. In the serum system the scavenge rate for InsP3 A . InsP3 B and InsP3 C were 91.78%, 84.18%, 91.18%,respectively.The antioxidant capacity of InsP3 could be ascribed to their coordination ability with metals.At higher concentration(20mg/mL ), InsP3 promoted hemolysis of mice red blood cell, but at lower concentration(2mg/mL ) it inhibited hemolysis of mice red blood cell. InsP3A and InsP3C demonstrated higher capacity in both promotion and inhibition abilities.After injection of InsP3 (ip.)100mg/kg.d for 12 days, The anti- ROS unit of mice serum was raised compared with the control group. There was a significant difference (P<0.01) between the controlled group and the intra-peritoneal injection of InsP3CInsP3 had no obvious effect on malondialdehyde (MDA) value in mice liver homogenate in vivo, but showed promotion effect on MDA formation induced by H2O2.0.04% InsP3C demonstrated better antioxidant activity than 0.02%BHT in oil system.The antioxidant mechanism of InsP3 is complex, especially in life system. Maybe their antioxidant activities were fulfiled through different mechanisms.5 Reduction effect on the level of blood glucose in diabetic miceCompared with the diabetic mellitus model control group induced by Alloxan, the levels of blood glucose in InsP3 groups were markedly reduced. The group injected with InsP3B acted the best in reducing blood glucose. When the concentrations of InsP3B were about 50mg/Kgd, and 100mg/Kg-d, the reduction rates of blood glucose were 13.34 %and 30.89%,respectively. Compared with the model control group, the damages of liver and kidney in InsP3 group were mitigated.InsP3 had the ability to raise the thymus indexes and spleen indexes of diabetic mellitus mice and normal mice. The InsP3B acted the best in this context. The capacity of reducing blood glucose of InsP3 may have relation with their improvement of immune function and antioxidant activity.6 Inhibition the growth of S180 tumor by InsP3Phytic acid and InsP3 prepared by 3 different ways could inhibit the growth of Sigo markedly, and acted in a dose-dependant manner when the injection concentrations were 150 mg/kg.d. , 100 mg/kg.d. -. 50mg/kg.d. The sequence of inhibition capacity of S-180 was as followings: InsP3C>phytic acid> InsP3B > InsP3A. At higher(150 mg/kg.d) concentration! the inhibition rate for InsP3A,InsP3B,InsP3C and InsP6 are 30.23%, 32.56%. 52.32%, 34.88%, respectively.InsP3 could increase the immune organ weight and promoted delayed-type hypersensitivity of Siso-bearing mice. InsP3 could effectively improve the content of serum hemolysin IgM and IgG in which they acted best at 100mg/kg.d. InsP3 could also increase the splenic antibody formation in tumor-bearing mice in a dose-dependent manner. When the tumor-bearing mice were treated with 150mg/kg.d InsP3, the splenic antibody in the tumor-bearing mice was near or even above the normal control group, and the InsP3A group acted the best.InsP3 significantly increased the activity of red blood cell catalase of tumor-bearing mice in a dose-dependent manner, the higher dose was more effective, InsP3B was most effective in higher concentration and phytic acid the least.Pathology observation of tumour tissue indicated that tumor cell necrosis andinvasion of inflammation did not play an important role in InsP3's anti-cancer activity. The mechanism may have something to do with its antioxidant activities and increasing organ immune capability.7 Inhibition effect on human hepatocarcinoma cell line SMMC-7721InsP3B displayed evidently growth inhibitory effect in a dose-and time-dependant manner against human hepatocellular carcinoma cell line SMMC-7721 byMTTtest. The tumor cell growth inhibitory rate after 72h treatment of 700 u g/mL InsP3B was 79. 08%. The result of PCNA test was in agreement with MTTtest.Both AFP test and morphology observation indicated that InsP3B might induce the differentiation of human hepatocellular carcinoma cell line SMMC-7721 to realize the inhibitory effect on the tumor cells.Flow cytometry analysis showed-that InsP3B could arrest the cells in G0/Gi phase which might induce the differentiation of tumor cells and therefore inhibited the proliferation of human hepatocellular carcinoma cell line SMMC-7721 in vitro.