Determination of protodioscin in tribulus terrestris by reversed-phase high-performance liquid chromatography

In this study, high performance liquid chromatography (HPLC) was used to analyze the protodioscin content in Tribulus terrestris samples. They were separated by C18 chromatography column, gradient with 0.1 % phosphoric acid solution and acetonitrile was used as the mobile phase with the flow rate of 0.5 mL/min, the column temperature was 45 °C, and the detection wavelength was 203 nm. The results showed that the protodioscin content has a good linear correlation (r>0.999) within the range of 50 mg/L to 1500 mg/L. In samples of raw material from Crimea it was found: 0.546 % ± 0.013 protodioscin and in analyzed material from Western Siberia it was 0.338% ± 0.008 for n=3. Raw materials of Tribulus terrestris growing in Crimea are more promising than similar raw materials from the West Siberian region due to the higher content of protodioscin for the production of biologically active products containing steroid saponins.


Introduction
Tribulus terrestris is an annual plant in the caltrop family (Zygophyllaceae). It grows in South Asia, Africa, Europe, northern Australia, Russian Federation (Crimean Republic, North Caucasus, Western and Eastern Siberia. Tribulus terrestris infusions, decoctions are widely used in alternative medicine in many countries. Bulgaria -erectile dysfunction treatment, Iran as a diuretic, laxative and syphilis treatment, India -radiculitis, in traditional Chinese medicine -eye diseases treatment [1]. In science-based medicine Tribulus terrestris extract is used as a booster for reproductive system. It is based on significant concentration of furostanol-type steroidal saponins in the plant represented by dioscin, protodioscin, tribestin, prototribestin, methylprototribestin, methylprotodioscin, pseudoprotodioscin, tribulosin, tribulusamide C and other compounds [2]. High concentrations of flavonoids, amino acids, lipids are also represented in Tribulus terrestris [3][4][5][6].
On Figure 1, the biologically active compounds in Tribulus terrestris chemical structure is presented.

Protodioscin Prototribestin
Dioscin Rutin Nutritional supplements with Tribulus terrestris extract are widespread among people involved in physical education and sport [11]. Plants bioactive substances helps to rise testosterone production. It is used as testosterone booster Saponins bind to the hypothalamus receptors. The hypothalamus signals the start of luteinizing hormone production [12].
Studies was made by panel scientists headed by Kazys Milasius. It showed that Tribulus terrestris supplements usage significantly helps to double or triple testosterone level due to the 8-20 days course of administration [13]. The same study found that Tribulus terrestris consumption result certain increase of strenght and aerobics activities data within sportsmens.
There are sources that illustrating the need for additional research on the Tribulus terrestris bioactive substances impact upon sportsmen physical efficiency [14].
S.G. Angelova, Z. Gospodinova, M. Krasteva и G.G. Antov illustrates Tribulus terrestris cancer protection abilities. It was set that general bulgarian Tribulus terrestris extract has a pronounced dose-related and abscopal effect on breast cancellation viability. Saponin fraction has an increased inhibitory effect compared to the total extract. Apoptotic bodies are involved in Tribulus terrestris anticancer activity mechanisms. Structural changes and DNA fragmentation were observed as markers of early and late apoptosis in tumor cells after treatment [15].
The study is intended to define protodoiscin in Tribulus terrestris through reversedphase high-performance liquid chromatography. Protodioscin standard was acquired in Sigma-Aldrich (G0299, CAS 55056-80-9). Acetonitrile was of HPLC grade and purchased from Lab-Scan (Poland).

Sample Preparation
Exactly 1.2 gram of the finely powdered plant material was extracted with 10 mL of 90% aqueous methanol by sonication within 60 min. After one day of infusion, the extracts were centrifugation and filtered through a 0.2-µm Iso-Disk Nylon filter (Supelco). Every sample solution was injected in triplicate.

Calibration
Standard compounds protodioscin (3 mg) was dissolved in 2.00 mL of 50% aqueous methanol (stock solution); further calibration levels were prepared by diluting the stock solution with the same water/methanol mixture. The resulting range for the concentration of protodioscin was from 50 to 1500.0 mg/L. All calibration levels were injected in triplicate, and the calibration data obtained (R2=0.9999 and y=1.379X+5.34) indicated linearity of the detector response in the range already mentioned.
Analytical Method. HPLC analysis was performed on a Agilent 1260 Infinity II LC system (equipped with a quaternary pump VL (G7111A), a vialsampler (G7129A), a multicolumn thermostat (G7116A), and a diode array detector VL (G1315D) from Agilent Technologies, Inc., Germany. A UCT Selectra AQUEOUS C18 column (150mm-2.1 mm, 5 µm particle size) from United Chemical Technologies (UCT) was used for all separations. The mobile phase, which consisted of water with of 0.1% phosphoric acid (A) and acetonitrile (B), was used for gradient elution from 90A/10B to 100B in 10 min, then to 100B in 2.5 min. The flow rate was adjusted to 0.5 mL/min, the detection wavelength was 203 nm, and 2 µL of sample was injected. All separations were performed at 45°С temperature. Peaks were assigned by spiking the samples with standard compounds and comparing retention times.

Results and discussions
Despite the fact that protodioscin is not very well absorbed in the ultraviolet spectral range, thanks to the ethane nitrile (acetonitrile) as a mobile phase, and the use of an analytical column with a small diameter, it was possible to create an analytical technique with a completely acceptable level of sensitivity for protodioscin (the minimum detection level is about 10 mg/L at a signal to noise ratio of 3/1). Protodioscin concentration dependence at the peak area is linear, with a high correlation coefficient (0.99992) as is shown in Figure 2.  Method was used to analyze protodioscin analysis in two plant samples. Protodioscin peak from sample chromatograms was identified with retention time as compared with standard chromatogram. In raw material samples from Crimea was found: 0.546 % ± 0.013 protodioscin and in analyzed material from Western Siberia it was 0.338% ± 0.008, for n=3 ( Figure 5). The data presented in Figure 5 indicate that the raw materials grown on the region of Crimea are promising for the production of phytopreparations. The data obtained are well consistent with those available in the literature [9,16].

Conclusions
With high performance liquid chromatography protodioscin identification in the raw plants a simple survey has been developed. It allows in 15 minutes determine the protodioscin contents and other steroid compounds in the prepared plant extracts under conditions that they are purchased as standards. Protodioscin content in the Tribulus terrestris aerial parts in studied samples growing in the Crimea is on average 60% higher than in similar raw materials from the West Siberian region. It is possible through favorable agro-climatic conditions on the peninsula. This fact provided an excellent opportunity for biologically active products containing steroid saponins from Crimean raw materials the production.