Silibinin is a non-toxic herbal product that possesses anticancer properties in different types of cancers, both in-vivo and in-vitro. Silibinin compounds, besides inducing apoptosis, can target different signalling pathways factors in cells, including transcription factors, growth factors, cell survival factors, inflammatory cytokines, kinase proteins, and angiogenesis factors. These might result in an effective and selective killing of cancer cells. Studies have shown that Silibinin has different target effects in cells (25).
Silibinin absorption and metabolism has been studied in different investigations. In the bloodstream, Silibinin is more observed in a conjugated form. In several studies, after oral prescription to healthy volunteers, only 10 to 17% of Silibinin was observed as a non-conjugated form (26, 27). Silibinin plasma concentration peak was obtained in mice after 30 minutes of injection and in tissues in min 60 post-injection. Then, the plasma concentration was reduced with the half-life of 57-127 min. It is while the concentration of conjugated Silibinin tends to be reduced with the half-life of 45-94 min (26). An important issue regarding Silibinin metabolism is its fast purgation in both forms of free and conjugated. In a clinical trial, the half-life of Silibinin was demonstrated in both forms to be almost 6.3 hours. Because of fast metabolism; its plasma concentration is usually in nanomolar (nM) range, while in a few cases, it is in micromolar (µM) range (27).
One of the key reasons for choosing Silibinin in developing anticancer targeted nanoparticles is its low toxicity in humans. For instance, in two clinical trials, Silibinin phytosome, which is a commercial formulation of Silibinin, was orally prescribed to prostate cancer patients in a dose of 13g/day and for the average of 20 days or 20-25g/day for the average of 28 days. The results showed that blood Silibinin after one-hour post-injection was approximately 20 µM without any severe toxicity for these patients. The lethal dose for intravenous injection in 50% of cases was reported to be 400mg/kg for mice and 385mg/kg for rats while other studies reported a safe injection of the drug in relatively higher doses (27).
Nevertheless, there are some challenges in applying Silibinin as an anticancer drug. Its separation and purification from its herbal origin, its pharmaceutical issues, long time and high costs of epidemiological studies on this drug, anticancer properties of flavonoids due to their poor solubility, poor oral absorption, and extensive hepatic metabolism are some of these barriers (28). There are some approaches to overcome these challenges. For instance, enhancers like Piperine, amid alkaloid extracted from Piperaceae herbal family can be a solution (29). To improve the pharmaceutical properties of Silibinin, another approach is to use sustained nanoemulsion synthesis technology. In this method, lipophilic flavonoids can be made in the form of emulsions comprised of nanoparticles with very small sizes (˂200nm). In this manner, flavonoids are gradually released and will have more absorption and biocompatibility after oral prescription (30).
An important issue in pharmaceutical research is to develop a precise, accurate, and repeatable method to quantify a drug in its specific solution in order to control the quality and assess its environmental sustainability, release from specific formulation, and pharmaceutical researches (31). To quantify a drug in solutions, a common way is chromatography. HPLC is a proper method to separate, measure, and demonstrate a type of material. It is one of the most common methods to quantify drugs in pharmaceutical researches. A great merit of this method is the opportunity to determine the structure and the level of impurity in drugs and formulations. This opportunity is not limited to synthetic drugs and can be applied to herbal drugs, too (32, 33).
This study was carried out to establish an experimental process using HPLC, based on previously-determined parameters in HPLC, such as choosing stationary, mobile phase and a detector, demonstrating the rate of mobile phase was utilized. Then, according to FDA guidelines, validation of a developed method, such as evaluation of a linear equation, validity and accuracy of findings, took place. Analysis of calibration curve in concentrations of 10, 20, 40, 60, 80, and 100 µg/ml suggested a linear correlation between concentration and peaks’ height. According to Figure 1, R2 in aqueous samples was 0.996, which reveals the linear connection between different concentrations. Retention time in in-vitro solution was 2.97 min that suggested a fast method for the identification of drug in in-vitro solutions. Analysis of inter-day and intra-day data (RSD ˂10.9%, precision 88%-105.9%) showed that the accuracy and precision of the developed method in the quantification of Silibinin in in-vitro solutions were acceptable.