| Macrothelypteris torresiana (Gaud.) Ching, which belongs to Thelypteridaceae family, is widely distributed in the south of China and has been used as folk medicine mainly for the treatment of diseases such as hydropsy and traumatic bleeding. In previous work, we have found that the total flavonoid fraction shows significant antitumor potential. In the total flavonoid fraction, the percentage of the protoapigenone, which is the main component, reaches up to 45%. In order to further investigate the antitumor activities of the total flavonoid fraction and obtain more data to explore it as a natural antitumor agent, The present study had the following objectives:(a) to establish the quality standard of Macrothelypteris torresiana and the total flavonoid fraction; (b) to optimize the inclusion process of the total flavonoid fraction by hydroxypropyl-β-cyclodextrin; (c) to compare and evaluate the acute and subacute toxicities, in vivo antitumor activities and pharmacokinetics of the total flavonoid fraction dissolved using sodium carboxymethyl cellulose (CMC-Na) and included by hydroxypropyl-β-cyclodextrin, respectively.The contents of water, ashes, acid insoluble ashes of powdered material of Macrothelypteris torresiana were determined according to the standard methods recorded in Pharmacopoeia of the People's Republic of China (AppendixⅨK and IX H in Part one of the 2005 version). We treated the protoapigenone as the reference substance and used the Ultraviolet-visible spectrophotometry method (Appendix VA in Part one of the 2005 version) to determine the contents of total flavonoids in powdered material of Macrothelypteris torresiana. Also, a HPLC method was used to determine the content of protoapigenone. As a result, the limits of the content of water, ashes, and acid insoluble ashes in the powdered material of Macrothelypteris torresiana were 15.01%,8.31% and 3.90%. By the statistical analysis on the above data with a Student's t-test, there was no difference among different places of origin (p>0.05). In the powdered material of Macrothelypteris torresiana, the contents of total flavonoids were equal to or higher than 3.31%, and the minimum content of protoapigenone was not less than 0.84%; the minimum content of protoapigenone in total flavonoids extrcts was not less than 37.06% Additionally, by the statistical analysis on the content of protoapigenone with a Student's t-test, there were some difference among different places of origin (0.010.05). But, the HE staining results showed that there was granular degeneration of liver cells only in the high dose group, additionally, preparation 1 and 2 showed the same results.The vivo antitumor investigation was performed on mouse sarcoma S-180 cells treated mice. The aqueous solution and suspension using CMC-Na of the total flavonoid fraction were administered orally at high, medium, low doses of 60,30, and 15 mg kg-1 body wt./day; inclusion of the total flavonoid fraction by hydroxypropyl-β-cyclodextrin at the doses of 40,20, and 10 mg kg-1 body wt./day; the standard drug 5-fluorouracil (20 mg kg-1 body wt./day) was administered orally to the positive control group. Sterile saline was administered orally in the same volume to the negative control group. The administration lasted for 10 days. The results showed that the inhibition ratios of tumor growth in suspension (35.23-56.35%) and inclusion (25.45-54.57%) groups were high than that of the aqueous solution group (<33.14%). the inhibition ratios of tumor growth in high and medium dose groups of both suspension and inclusion were almost the same (p>0.05), but the low dose group did not show enough high activities (p<0.05). Compared with positive control, there is no difference of the high and medium doses of both suspension and inclusion (p>0.05), but significant differences of all doses of the aqueous solution (p<0.05).In this part, the pharmacokinetics of the suspension using CMC-Na and inclusion by hydroxypropyl-β-cyclodextrin of the total flavonoid fraction was performed. Doses (100,200,300 mg kg-1) of the suspension and inclusion were administered orally to rats, the two compartment model and noncompartment model were used to analyze protoapigenone in plasma by Das 2.0. We found that the two models both showed a significant linear kinetics in rats, and the AUC was proportional to the dose. Additionally, besides the half-life time had nothing to do with the dose, the two models had the same kinetics process. Anymore, the inclusion extended the half-life time, shortened peak time, and increased peak concentration. After administion, the protoapigenone from the total flavonoid fraction was mainly distributed in liver, kidney, lung, spleen and heart, which are organs with abundant blood flow, and some even distributed in breast, ovarian and uterine issues, but less distributed in brain, muscle and fat issues. In bile, urine and feces, we found certain amount of protoapigenone excreted as prototype, and compared the amount excreted, urine> feces> bile. After inclusion by hydroxypropyl-β-cyclodextrin, the excretion of the protoapigenone in feces was significantly reduced, thus the inclusion might promote the gastrointestinal absorption of protoapigenone. In all plasma and organs, there is a common metabolite, which might be formed from the degradation of protoapigenone, but it requires further in-depth studyThe equilibrium dialysis combined with HPLC was carried out for the determination of the plasma protein binding rate of protoapigenone. From the results, we found that the balance time of dialysis was 10 h in human plasma, and the plasma protein binding rates of protoapigenone at low, middle and high concentrations (200,500,1000μg L-1) were 90.5±1.2%,89.6±1.7% and 91.4±1.4%, respectively. But the balance time of dialysis was 6 h in rat plasma, and the plasma protein binding rates of protoapigenone at low, middle and high concentrations were 80.7±2.2%,81.1±1.8% and 81.9±1.3%, respectively. Thus, the plasma protein binding rates of protoapigenone in both human and rat plasma were high enough. Additionally, there are significant differences between the plasma protein binding rates of protoapigenone in human and rat plasma (p<0.01), but no difference in low, middle or high concentrations (p>0.05). |
PDF Full Text Request