| Tripterygium wilfordii Hook.f.(TWHF) is a traditionnal Chinese medicinal herb. It has been widely used in the clinical treatment of rheumatoid arthritis,other inflammatory and autoimmune diseases and male fertility control.Pharmacology studies have shown that it has immunosuppressive activity,anti-tumor and anti-inflammatory effect. However,TWHY have big poison,its toxicity from its complex chemical composition and physiological active ingredients,its toxicity size is two terpenoids,alkaloids, triterpene and glycosides. Triterygium wilfordii clinically therapeutic window is narrow,the main active ingredients such as triptolide and tripterine are both active ingredients and toxic ingredients. Therefore,in order to safe and effective clinical application,is crucial to find out the effective components from TWHY and its effective dose and toxic dose. In this paper,we mainly research on the following aspects.1. The toxicity of TWHY monomers and preparations to the HL-7702cellsDue to the side effects of tripterygium wilfordii is harmful for multiple system, particularly for the liver system. HL-7702cells are normal liver cells, which is a good cell model to study drugs’function and mechanism on live cell in vitro. So we choose this cell line to identify the toxic components in TWHY. This study choose eight kinds of monomers which were isolated by teacher. triptolide and triptophenolide belongs to two terpenoids, tripterine and wilforlide A belongs to triterpene, wilforidine、wilforgine、wilforine and wilfordine belongs to alkaloids. Tripterygium wilfordii preparations are LGT tablets and LGTDG tablets.To study the triptergium wilfordii monomers and preparations on the HL-7702cell toxicity effect by measuring cell proliferation inhibition rate. The8kinds monomers and66batches preparations of TWHY were acted on HL-7702cells for24h in different concentrations respectively. MTT colorimetric method was used to determine the proliferation inhibition rate of HL-7702cells.The result found that wilforidine, wilforgine, wilforine, wilfordine and triptophenolide in200μg/ml have no inhibitory effect on the HL-7702cells. The IC50of wilforlide A was greater than200μg/ml, the IC50of triptolide was199.7μg/ml, the inhibitory of triptolide have no clear concentration-response relationship.the IC50of tripterine was3.56μg/ml. The result explained that wilforidine, wilforgine, wilforine, wilfordine and triptophenolide without toxicity, the other3monomer toxicity level in descending order are tripterine, triptolide, wilforlide A.The preparations were characterized by its effect on promoting cell growth in low concentration group,showed different inhibition in moderate group,In the high concentration group were characterized by high intensity of inhibition, which may be associated with certain toxicity component content in the preparation.2. Screening immunosuppressive activity monomers from TWHYTo screen immunosuppressive activity monomers from TWHY by using mouse spleen lymphocyte transformation test in vitro.8kinds of monomers and66batches preparations of TWHY respectively in different concentration have acted on mice spleen lymphocytes (which stimulated by ConA and LPS)for48h in vitro. MTT colorimetric method was used to determine the inhibition rate of lymphocytes cells which were stimulated by ConA and LPS.The result found that wilforidine, wilforgine, wilforine, wilfordine and triptophenolide in100ug/ml have no inhibitory effect on the stimulated lymphocytes cells. The IC50of triptolide that stimulated by ConA and LPS were2.34ng/ml,1.24ng/ml respectively. The IC50of tripterine that stimulated by ConA and LPS were87.43ng/ml,36.10ng/ml respectively. The IC50of wilforlide A that stimulated by ConA and LPS were1.29mg/ml,0.87mg/ml respectively. The result explained that wilforidine, wilforgine, wilforine, wilfordine and triptophenolide without immunosuppressive activity, the other3monomer immunosuppressive activity level in descending order are triptolide, tripterine, wilforlide A.The results of Tripterygium preparations showed that:most of samples in different concentrations perfoemanced diffenent inhibitions, the inhibitions between different enterprises and the same enterprise in different batches were different.3. Determination of tripterine in tripterygii preparationsPreliminary experiments have proved that the immunosuppression activity of tripterine is strong, but its toxicity is largest. At present, the quality control of tripterygium wilfordii wilfordii preparations without the content of tripterine,It is important to control the content of tripterine. using Welch UltimateTM XB-C18column to acetonitrile-0.1%phosphoric acid (80:20) as the mobile phase, the column temperature was25℃, and the flow rate was1mL·min-1, as well as detection wavelength was425nm. We developed an HPLC method for the determination of tripterine in LGT tablets and LGTDG tablets.The results found that the linear range of tripterine was0.472～94.4μg/ml, and the average recoveries were98.32%(RSD=3.13%) and101.19%(RSD=2.17%) in LGT tablets and LGTDG tablets respectively. The method is simple, accurate and reproducible. It can be applied to determine the contents of tripterine in tripterygii preparations.4. Determinatin of triptolide and wilforlide A in tripterygii preparationsPreliminary experiment shows that triptolide have stronger immunosuppressive activity, lower toxicity than tripterine. The toxicity of wilforlide A is relatively small, but its effective dose is larger. The present quality standard of Tripterygium wilfordii preparation is mainly to control these2active ingredient content. In order to evaluate the relationship between these two ingredients and cell inhibition rate,we need to determinate the content of triptolide and wilforlide A of tripterygii preparations.5. The correlation analysis between chemical composition and the inhibition rate of HL-7702cellsTripterygium wilfordii wilfordii is a traditional Chinese herbal. due to the complexity composition, its therapeutic effect is usually a result of the combined action of the chemical compositions,as well as the toxicity effect. In order to clarify the relationship of tripterine, triptolide and wilforlide A with cell toxicity,we analysis these three components’ content and inhibitory rate.The result found that the preparations were characterized by its effect on promoting cell growth in low concentration group, showed different inhibition in moderate and high concentration group. the concentration of tripterine and wilforlide A were correlated with cell inhibition rate. In the monomer test, the toxicity of wilforlide A is smallest, but in the preparations test, the content of wilforlide A is most relevant than other two monomers.The result shows that the main components of cytotoxicity induced by TWHY is triptolide and tripterine. Lt prompt that in Tripterygium wilfordii preparation,there are some chemical compositions interacted with wilforlide A, and make it morer sensitivly. |
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