Preclinical Study Of The Anti-urolithiatic New Drug Of Alismatis Rhizoma

Objective: To carry out the preliminary preclinical study of the anti-urolithiatic active constituents of Alismatis Rhizoma basing on previous studies. First, to confirm the anti-urolithiatic active fraction of Alismatis Rhizoma under further study and to optimize the preparation process of the active constituents. Second, to confirm the anti-urolithiatic active constituents of Alismatis Rhizoma through pharmacology study. Third, to establish the quality standard of the extract of the anti-urolithiatic active constituents of Alismatis Rhizoma.Methods and Results:1. Extracorporeal pharmacology study was used to confirm the anti-urolithiatic active fraction of Alismatis Rhizoma. Different extracts were prepared, including the total triterpenoids extract (TTE), the extracts of ethyl acetate, petroleum ether, n-butanol and water, crude extracts of volatile oils and alkaloids. The inhibitory effects of different extracts were evaluated in vitro using a standard seeded crystallization method. The results showed that TTE and the ethyl acetate extract had strong inhibition on the formation of CaOx crystal in vitro with the inhibitory index of 124.50% and 121.53%, respectively. There were significant differences when compared to the normal control. The results illustrated that the ethyl acetate extract was the anti-urolithiatic active fraction of Alismatis Rhizoma and that the total triterpenoids were the anti-urolithiatic active constituents of Alismatis Rhizoma.2. The HPLC-ELSD determination of Alisol B 23-acetate was optimized. 65% acetonitrile was used as mobile phase. The methodology examination showed that the calibration curve was linear in the range of 0.544～2.720μg. The precision RSD was 2.10% and the average recovery was 101.34%. The method could be used in the determination of Alisol B 23-acetate. Then the extract efficiency on total triterpenoids and the evaluation index Alisol B 23-acetate of different concentrations of alcohol were investigated. The results showed that 60% alcohol could extract more total triterpenoids and Alisol B 23-acetate. And 60% alcohol was used to optimize the preparation process of the active constituents. 3. Ethylene glycol and alfacalcidol were used to induce CaOx urinary stone model in male SD rats. The prophylactic effect of TTE on urolithiasis was evaluated by determining the serum biochemical, urinary and renal parameters. Kidney sections were also examined under light microscope to study the CaOx crystal deposition and pathological changes. The results showed that the administration of TTE decreased the levels of serum urea and creatinine, urine oxalate and Ca²⁺, relative kidney weight and renal Ca²⁺ in the urolithiasis SD rats. Also, TTE increased the renal Mg²⁺and 24h urine volume. It also diminished the number and size of CaOx crystal deposits and protected the kidneys from serious damage. The results illustrated that TTE could inhibit the formation of urinary stone in rats.4. To establish the quality standard of the TTE of Alismatis Rhizoma according to the Pharmacopoeia of the People's Republic of China (2005), VolumeⅠ, including identification, examination, assaying, et al. According to the determination results of several batches of TTE, the standard was set as follows: The contents of the total triterpenoids and Alisol B 23-acetate should be more than 55% and 1.10%, respectively. The water content and the ignited residue content should be within 9% and 0.5%, respectively. The content of heavy metal and arsenic salt should be within 20ppm and 2ppm, respectively.Conclusions: TTE has a beneficial effect against CaOx urinary stone formation induced by EG and alfacalcidol. The total triterpenoids are the anti-urolithiatic active constituents of Alismatis Rhizoma and may be useful in the treatment of urolithiasis, especially in the prevention of recurrence. However, further pharmacological studies are required to confirm more comprehensive anti-urolithiatic mechanisms.