PHARMACOKINETIC STUDIES OF TRIBESTAN

N. Dikova, V. Ognyanova

Chemical Pharmaceutical Research Institute
Sofia, Bulgaria

Tribestan is a natural product of plant origin. It has been extracted from the above-the-ground part of Tribulus terrestris L., containing mainly steroid glycosides (saponins) .of furostanol type with predominating quantity of protodioscine [1, 3, 11, 12, 13]. Tribestan has been standardized on the basis of furostanol saponins (not less then 45%) [1]. Its administration to humans and animals improves the sexual libido and stimulates spermatogenesis [2].

The objective of the present work is to study the pharmacokinetic behavior of Tribestan substance in experimental animals.

MATERIALS AND METHODS

The pharmacokinetic characterization of Tribestan was performed with respect to the uncharged protodioscine, the predominant component of the product.

Experiments were carried out on 265 male Wistar albino rats with body weight from 160 to 270g, depending on the requirements of the experiment, to determine the plasma concentrations and excretion of the preparation.

The protodioscine plasma concentrations were determined in a group of 50 rats (160-180 g), treated intravenously with a single dose of Tribestan substance, and a second group of 100 rats treated intravenously with a dose of 200 mg/kg. Citrate blood was withdrawn after decapitation of the experimental animals 2, 4, 10, 20, 30, 45, 60, 90, 120 and 180 minutes after the treatment.

For the determination of protodioscine excretion in the bile, 60 rats (230-270 g) were treated with a single intravenous dose of 50 and 200 mg/kg, and 30 rats (230-270 g) were treated orally with a single dose of the same quantities of Tribestan substance. The rats were narcotized by intraperitoneal administration of 40-mg/kg chloralose, 100% urethane and their biliary ducts were cannulated. Bile was dynamically followed up until the 6th hour, 6-9 h, and 9-20 h after a single intake.

For the determination of protodioscine excretion with the urine, 25 rats (160-180 g) were treated intravenously and orally with Tribestan in doses of 50 and 200 mg/kg. The rats were left in metabolic cages for separate collection of urine and feces. Urine was collected for 24 h.
All experimental animals were treated after 16-hour fasting - the control animals were kept under identical conditions.

Tribestan was administered as 5 to 20% aqueous solutions, and all doses used were recalculated as 100% pure substance.

The determination of unchanged protodioscine in the plasma, bile and urine was performed by thin-layer chromatography, and the quantitative determinations were performed in comparison with certain protodioscine concentrations.

Plasma was processed as follows: 10 ml methyl alcohol was added to 1 ml from it. After stirring, it was left for 10 minutes and then centrifuged. The solution was evaporated to dry over a vacuum evaporator and the dry residue, dissolved in 0.4 ml methanol, was streaked upon the chromatographic plate.

Bile and urine were applied without previous treatment (native) in proper quantities (0.02 to 0.06 ml).
Thin-layer chromatography was applied on carrier Kieselgel LG and mobile phase n-butanol; acetic acid; water (4:1:2:2), when protodioscine gave only one spot with Rf = 0.50 [4, 7, 8, 9, 10, 11]. Ehrlich's reagent was used as developer. Detectable minimum 1 mkg per sample in concentration range from 1 to 20 mkg and the reproductivity of the method was about 70%.

RESULTS AND DISCUSSION

The results of the determination of the plasma concentrations of protodioscine, after intravenous administration of both Tribestan doses, are illustrated in Fig.1 (Diagram 1).

The curves, representing the change of protodioscine concentrations in time, have a marked steep course during the first ten minutes after the treatment. The profile of the concentration curves indicates that protodioscine is rapidly eliminated from the plasma and by 180 min its concentration in negligible.

After oral administration of Tribestan, no measurable concentrations of protodioscine were found.
The experiments for studies of Tribestan excretion reveal that about 12 and 14 % of the component is excreted in the bile within 24 hours, and in the urine - about 6 to 7% protodioscine, compared to the doses of 50 and 200 mg/kg respectively, administered as a single intravenously. After oral administration of Tribestan in the same doses, a smaller quantity of protodioscine was excreted in the bile of rats within 24 hours - from 2 to 4% depending on the dose. No measurable concentrations of unchanged protodioscine were established in 24-hour urine and feces of rats,

Tribestan-treated with oral doses of 50 and 200 mg/kg.

The experimental data obtained indicate that the hepatic route is the route of choice for Tribestan excretion as unchanged protodioscine. It can be assumed that protodioscine, being the predominating part of the active component of the product, participates in the enterohepatic cycle of rats. This hypothesis is based on experimental data from Tribestan excretion as unchanged protodioscine after intravenous and oral administration, as well as on the chemical similarity of the product studied with some already known pharmaceuticals, whose participation in the enterohepatic cycle in experimental animals has been confirmed [5, 6].

The rapid elimination of protodioscine from the plasma, as well as low percentages of unchanged protodioscine excreted versus the administered dose, support the opinion that protodioscine undergoes intestinal biotransformation in the body. Actually, the presence of other chromatographic spots, different from that of unchanged protodioscine, have been identified during thin-layer chromatographic determination of the samples of urine and bile (according to Rf values). The problem of Tribestan biotransformation needs further research work.

REFERENCES

1. Tomova M., Gyulemetova R., Zarkova S. Patent No 27 584 A61 (in Bulgarian).
2. Tomova M., Gyulemetova R., Zarkova S., Peeva S., Pangarova T., Simona M. Steroid saponins in Tribulus terrestris L. with action stimulating sexual function. Report at the First International Conference of Chemistry and Biotechnology of Bioactive Natural products. Varna, Bulgaria, September 21-26, 1986 (in Bulgarian).
3. Tomova M., Panova D. Steroid sapogenins. Isolation of diosgenin from Tribulus terrestris L. Farmazia, 4, 211, 1965 (in Bulgarian).
4. Ehwanajje D., Layer M., Radwan A. Steroid sapogenins. IX. Thin-layer chromatography. Phytochemistry, 4, 587, 1965.
5. Gayen M., Dwornik D. Effect of diosgenin on lipid metabolism in rats. J. Lipid. Res., 20, 162, 1979.
6. Gayen M., Jerdin E., Graselin E., Dwornik D., Studies on the disposition of diosgenin in rats, dogs, monkeys and man. Atherosclerosis, 33, 71, 1979.
7. Kanasaki T., Miyahara K. Thin-layer chromatography of steroid saponins and their derivatives. Chem. Pharm. Bull., 11, 1546, 1963.
8. Kiyosawa S., Huton M. Detection of prototype compounds of Diosgenin and other spirostanol glucosides. Chem. Pharm. Bull., 16, 1162, 1968.
9. Raymond D., Heftmann E. Thin-layer chromatography of steroidal sapogenins. J. Chromatography, 9, 353, 1962.
10. Takeda K., Hara S., Wada A., Matsumoto N. A systematic simultaneous analysis of steroid sapogenins by thin-layer chromatography. J. Chromatography, 11, 562, 1965.
11. Tomova M., Botscheva D., Zaikin W., Wulfson N. Steroidsaponine und Steroidsapogenine v. Hepogenin aus Tribulus terrestris L., Planta medica, 3, 223, 1977.
12. Tomova M., Panova D., Wulfson N. Steroidsaponines and steroidsapogenines. IV. Saponins from Tribulus terrestris L. Planta medica, 25, 231, 1974.
13. Tomova M., Gjulemetova R. Steroidsaponines and steroid sapogenines. IV. Saponins from Tribulus terrestris L. Planta medica, 34, 188, 1978.
14. Tschesche R., Wulf G. Konstitution und Eigenschaften der Saponine. Planta medica, 3, 274, 1964.