Preparation And Evaluation Of Standardized Total Lignans Of Herpetospermum Caudigerum Nanosuspensions

Most of active components extracted from the traditional Chinese medicine showed good biological activity in treatment of corresponding diseases, many of them belonged to the bioavailability classification system class II drugs with low dissolution rate and solubility. This was not only to reduce the clinical curative effect and increase adverse reactions, but also lead to high failure probability, high risk and high investment of new drug development. Nanosuspensions, as a new drug delivery system for poorly water-soluble drugs, can effectively improve the oral bioavailability of drug and has been a research hotspot in pharmaceutical field.Modern pharmacological researches has demonstrated that total lignans of Herpetospermum caudigerum (TLHC) possessed a variety of biological activities, such as anti-hepatitis B virus, inhibiting experimental hepatic injury, anti-HBV. However, TLHC was poorly water-soluble active extractions with low oral bioavailability, which seriously limited its clinical application. Most of active components extracted from TLHC contained multiple phenol hydroxyls in the chemical structure, so its solubility behavior was easily affected by pH value. Therefore, our study tried to utilize dissolving-precipitating method based on acid-base neutralization reaction to produce TLHC nanosuspensions for improving its solubility and oral bioavailability, which would provide a new and effective method to solve the poorly water-soluble problem of active extractions with weakly acidity or alkalinity extracted from the traditional Chinese medicine.In this paper, total lignans of Herpetospermum caudigerum nanosuspensions (TLHC-Ns) was prepared by precipitation-combined homogenization technology based on acid-base neutralization reactions, and the critical factors involved in the preparation prooess and formulation composition like the type and concentration of stabilizers, drug concentration, homogenization pressure and cycles were optimized through the single factor investigation or Box-Behnken design-response surface method. The optimal preparation process was as follows:TLHC 1.5g (1.5%, w/v) firstly was dissolved in a 10 mL mixed solvent with 5 mL 0.1 mol/L NaOH and 5 mL ethanol by ultrasonic method, and then slowly dropwised into 95 mL aqueous solution containing 0.21 g SDS (0.21%, w/v),0.33g PVP K30 (0.33%, w/v) and 5 mL 0.1 mol/L HCl under 1000 rpm stirring. A flavescent suspension spontaneously formed with gradually adding alkaline droplets containing TLHC. The freshly prepared suspension was further homogenized under homogenization process of 500 bar for the first 5 circles and 1200 bar for the next 15 circles using a piston-gap high pressure homogenizer to produce uniform TLHC-Ns with small particle size. Under the condition of optimized process and formulation, prepared TLHC-Ns had about 300 nm of particle size and 0.141 ofpolydispersity (PDI).Because of poor physical stability of TLHC-Ns, it is necessary to further transform it into solid form. Optimal dried TLHC-Ns with 345 nm of particle size and 0.187 of PDI was prepared by lyophilization, and 4%(w/v) mannitol was selected as cryoprotectants to facilitate freeze-drying preparations easily reconstitute to original nanosuspensions. From the scanning electron microscopy (SEM) image, dried TLHC-Ns were composed of individual near-spherical-shaped particles which were split into a single by the rod-like mannitol. For XRD of optimal dried TLHC-Ns, TLHC was exhibited in amorphous form, and didn't appear crystalline transformation during the process of preparation and solidification of TLHC-Ns. Contrast to raw drug, dissolution rate of dried TLHC-Ns was significantly higher in shorter time. Besides, Short-term stability test showed dried TLHC-Ns possessed higher stability at both room temperature and 4? refrigerator during 7 weeks, in comparasion with TLHC-Ns.In this paper, our study adopted common acute liver injury model of rats induced by intraperitoneal injection of carbon tetrachloride (CCl4) to evaluate hepatoprotective effects of dried TLHC-Ns. As the result shown, each dose group can effectively reduce the levels of ALT, AST and MDA and pathological changes of liver cell degeneration and inflammatory infiltration, and improve the activities of SOD in a dose-dependent manner. However, hepatoprotective effects of dried TLHC-Ns were significantly higher than raw drug at the same dose, and efficacy of its high, middle dose group almost were close to that of bifendate (BDD). Besides, in vitro HepG2 2.2.15 cells model and in vivo duck hepatitis B virus (HBV) model were used to investigate the biological activity of anti-HBV of dried TLHC-Ns, and the results revealed similar efficacy. In the in vitro test, dried TLHC-Ns effectively suppressed the secretion of the HBV antigens (HBsAg and HBeAg) in a dose-dependent manner with significant difference from raw drug. In the in vivo evaluation, dried TLHC-Ns with high dose and middle dose significantly reduced the serum HBV DNA level, and the efficacy of dried TLHC-Ns was better than that of raw drug and was almost close to or more than that of lamivudine. Most important of all, rebound phenomenon didn't occur after dried TLHC-Ns or raw drug withdrawal.To sum up, the results showed that precipitation-combined homogenization techniques based on acid-base neutralization reaction was successfully exploited to prepare TLHC-Ns with small particle size, which could effectively improve dissolution and solubility of TLHC, and then increase clinical curative effects of TLHC. Therefore, nanosuspensions could be used as an ideal choice for oral delivery TLHC. Besides, this also set an example to solve the poorly water-soluble of problem of active extractions extracted from the traditional Chinese medicine with weakly acidity or alkalinity.