| According to Chinese medicine backward production technology, nonstandard quantitative method, and unstable curative effect, we successfully established a set of standardized biomolecular integrational technology in previous work. Application of the technology to separate and purify GuiZhi-Tang(GZT), the products were analyzed by HPLC finger-print. The results indicated that the components were similarity between biotransformation products and in rat portal vein afer oral administration of GZT. Basing on the above method, using enzyme-linked immunosorbent assay (ELISA) and Western blotting (Western blot) methods, we observed the effects of Guizhitang and it's biotransformation product on p 38 MAPK, cPLA?, SPLA2, COX, PGES,15-PGDH, NF-?B and PGE2 in bEnd.3 with IL-1?inducement, and to investigate the effects of GZT and it's biotransformation product on catabolizing enzymes of PGE2. We wish it can provide the evidence for this technology, and to improve TCM preparation forms, and creat new form drug.1 Preparation of GZT, serum containing of GZT and its biotransformation productsGZT was prepared with conventional method. The biotransformation product preparation route was optimized as follow:100 mL enzyme solution of intestinal flora enzyme (pH7.5) with protein concentration of 1300 mg·L-1 was added into 20 mL GZT with crude drug concentration of 1.0 g/mL. The solution was kept at 42?for 36 hours. After the reaction was completed, the solution was clarified by centrifugation at 7000 r·min-1 for 15 min. The clarified liquid was purified by ultra filtration with membrane pore size 500 000 dalton. The ultrafiltrate was concentrated by vacuum concentration to suitable density. Serum containing of GZT and its biotransformation products were prepared with conventional method. All of them were using for the following studies.2 Antipyretic effect of GZT on fever in ratsIt was found that body temperature was significantly high after 3.5 h induced by yeast. Rat temperature decreased significantly after GZT ig 2 hours. It showed that GZT can decrease rat body temperature significant, which effects of GZT low-dose group appeared on 2 h,3 h, GZT high-dose group on 1 h and 2 h (P<0.05). Aspirin group was significantly inhibited rat temperature induced by yeast after 1 h,2 h and 3h.After given yeast, compared with normal group, content of IL-1?,PGE2 in serum were significantly increased. Two groups of GZT and aspirin can significantly reduce the serum IL-1?, PGE2 content. It Showed that the yeast can induced body temperature increasing, and GZT can inhibited the effect by decreasing content of IL-1?and PGE2.3 Effect of GZT and it's biotransformation product on p-p 38 MAPK in bEnd.3 with IL-1?inducementAfter adding IL-1?in cells 10 h, compared with control group, there was no significant changes on expression of p 38 MAPK of bEnd.3, but p-p 38 MAPK protein expression increased significantly. Serum-containing-GZT, biotransformation products and serum-containing-biotransformation products can reduce the p 38 MAPK protein expression mediated by IL-1?. Serum-containing-GZT and serum-containing-biotransformation products showed concentration dependent trend, and it was not appeared on biotransformation products group.4% biotransformation products had the most significant effect. The results indicated that target site of GZT and it's biotransformation product is p-p 38 MAPK.4 Effect of GZT and it's biotransformation product on cPLA2, sPLA2 in bEnd.3 with IL-1?inducementELISA results suggested that cPLA2 and sPLA2 levels were significantly increased compared with control group after given IL-1?10 h(P<0.01). There were no significant difference on secretion of cPLA2 on 2.5% serum-containing-GZT group. And secretion of cPLA2, sPLA2 on 2.5% serum-containing-biotransformation products group showed non-significant difference either. The other concentrations groups can significantly reduce the extracellular fluid content of cPLA2 and sPLA2, and presented an obvious trend of concentration dependence. IC50 values on cPLA2 of serum-containing-GZT, serum-containing-biotransformation products, biotransfor-mation product were:10.199,11.468 g/kgBW/U and 52 g/L. IC50 values on cPLA2 were:6.909,7.379 g/kgBW/U and 44 g/LAfter adding IL-1?in cells 10 h, compared with the control group, cPLA2 protein expression increased significantly. Serum-containing-GZT, biotransformation product, serum-containing-biotransformation products can reduce the IL-1?-mediated cPLA2 protein expression. Groups of serum-containing-GZT and serum-containing-biotrans-formation products showed no gradient dependence. Effects of 2%,4% concentration groups were better than 1% serum-containing-biotransformation products group.5 Effect of GZT and it's biotransformation product on COX-2 in bEnd.3 with IL-1?inducementAfter adding IL-1(3 in cells 10 h, compared with the control group, COX-2 protein expression increased significantly. Serum-containing-GZT, biotransformation product, serum-containing-biotransformation products can reduce the IL-1?-mediated COX-2 protein expression. Serum-containing-GZT group showed obvious trend of concentration dependence. There was no significant differences on expression of COX-2 of 1% biotransformation products group, but had apparent effect of 2%.4% concentration groups. There was no significant differences on expression of COX-2 of 5%.10% serum-containing-biotransformation products groups, but had apparent effect of 20% concentration group.6 Effect of GZT and it's biotransformation product on PGES in bEnd.3 with IL-1?inducementELISA results suggest PGES content was significantly increased compared with control group after given IL-1?10 h. Serum-containing-GZT. biotransfor-mation product, serum-containing-biotransformation products can reduce the IL-1?-mediated PGES secretion, and presented an obvious trend of concentration dependence.There were no significant differences on secretion of PGES of 2.5% serum-containing-GZT group, and the other concentrations groups can significantly reduce the extracellular fluid content of PGES. There were no significant differences on secretion of PGES of 1% biotransformation products group, and the other concentrations groups can significantly reduce the extracellular fluid content of PGES. IC50 values on PGES of serum-containing-GZT, serum-containing-biotransformation products, biotransfor-mation product were:10.575,13.207 g/kg BW/U and 40 g/LAfter adding IL-1?in cells 10 h, compared with the control group. PGES protein expression increased significantly. Serum-containing-GZT, serum-containing-bio-transformation products can reduce the IL-1?-mediated PGES protein expression. There was no significant difference on biotransformation product.7 Effect of GZT and it's biotransformation product on 15-PGDH in bEnd.3 with IL-1?inducementELISA results suggest 15-PGDH content was significantly increased compared with control group after given IL-1?10 h. Serum-containing-GZT, biotransformation product, serum-containing-biotransformation products can reduce the IL-l(3-mediated 15-PGDH secretion, and presented an obvious effect relationship. Comparison of the efficacy curve found, serum-containing-GZT and biotransfonnation product had a similar effect on 15-PGDH. IC50 values on PGES of serum-containing-GZT, serum-containing-biotransformation products, biotransformation product were:8.554,10.434 g/kgBW/U and 45 g/LAfter adding IL-1?in cells 10 h, compared with the control group,15-PGDH protein expression increased significantly. Serum-containing-GZT group showed obvious uend of concentration dependence. There were no significant differences on expression of 15-PGDH of 1%.2% biotransformation products groups, but had apparent effect of 4% concentration group. Serum-containing-biotransformation products group showed obvious effect relationship.8 Effect of GZT and it's biotransformation product on NF-?B in bEnd.3 with IL-1?inducementAfter adding IL-1?in cells 10 h, compared with the control group, no significant changes appeared on expression of NF-?B, but p-NF-KB protein expression increased significantly. There were no significant differences on expression of low concentrations groups of serum-containing-GZT, biotransformation products and serum-containing-biotransformation products, but can reduce the IL-1?-mediated p-NF-?B protein expression. It indicated that target site of GZT and it's biotransformation product is p-NF-KB.9 Effect of GZT and it's biotransformation product on PGE2 in bEnd.3 with IL-1?inducementELISA results suggest PGE2 was significantly increased compared with control group after given IL-1?10 h. Serum-containing-GZT, biotransformation product, serum-containing-biotransformation products can reduce the IL-1?-mediated PGE2 secretion, and presented an obvious trend of concentration dependence. IC50 values on PGE2 of serum-containing-GZT, serum-containing-biotransformation products, biotransformation product were:8.601,10.528 g/kgBW/U and 56 g/L.The yeast can induced body temperature increasing, and GZT can inhibited it by decresed content of IL-1(3 and PGE2. GZT and it's biotransformation product can inhibit the changing on p 38 MAPK, cPLA2, SPLA2, COX-2, PGES,15-PGDH, NF-?B and PGE2 in bEnd.3 with IL-1?inducement. Intensity from strong to weak of biotransformation product is PGES(40)>sPLA2(44)>15-PGDH(45)>cPLA2(52)> PGE2 (56). Intensity from strong to weak of serum-containing-GZT is sPLA2 (6.909) >15-PGDH(8.554)>PGE2(8.601)>cPLA2(10.099)>PGES(10.575). Intensity from strong to weak of serum-containing-biotransformation product is SPLA2 (7.379)> 15-PGDH(10.434)>PGE2(10.528)>cPLA2(11.468)>PGES(13.207).The results sugest that there are equal effectiveness between GZT and it's biotransformation product. It can provide experimental evidence for biotrans-formation of TCM. |
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