Pharmacokinetic Analysis of Diosgenin in Rat Plasma by a UPLC-MS/MS Approach.

Diosgenin, a steroidal sapogenin, has attracted attention worldwide owing to its pharmacological properties, including antitumor, cardiovascular protective, hypolipidemic, and anti-inflammatory effects. The current diosgenin analysis methods have the disadvantages of long analysis time and low sensitivity. The aim of the present study was to establish an efficient, sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach for pharmacokinetic analysis of diosgenin amorphous solid dispersion (ASD) using tanshinone IIA as an internal standard (IS). Male Sprague-Dawley rats were orally administered diosgenin ASD, and orbital blood samples were collected for analysis. Protein precipitation was performed with methanol-acetonitrile (50 : 50, v/v), and the analytes were separated under isocratic elution by applying acetonitrile and 0.03% formic acid aqueous solution at a ratio of 80 : 20 as the mobile phase. MS with positive electron spray ionization in multiple reaction monitoring modes was applied to determine diosgenin and IS with m/z 415.2⟶271.2 and m/z 295.2⟶277.1, respectively. This approach showed a low limit of quantification of 0.5 ng/ml for diosgenin and could detect this molecule at a concentration range of 0.5 to 1,500 ng/ml (r = 0.99725). The approach was found to have intra- and inter-day precision values ranging from 1.42% to 6.91% and from 1.25% to 3.68%, respectively. Additionally, the method showed an accuracy of -6.54 to 4.71%. The recoveries of diosgenin and tanshinone IIA were 85.81-100.27% and 98.29%, respectively, with negligible matrix effects. Diosgenin and IS were stable under multiple storage conditions. Pharmacokinetic analysis showed that the C max and AUC0⟶t of diosgenin ASD were significantly higher than those of the bulk drug. A sensitive, simple, UPLC-MS/MS analysis approach was established and used for the pharmacokinetic analysis of diosgenin ASD in rats after oral administration.

The complete chloroplast genome of Atractylodes japonica Koidz. ex Kitam. and its phylogenetic inference.

Atractylodes japonica Koidz. ex Kitam. is a perennial herbal plant, and its dried rhizomes have been widely used as traditional medicine in China and Japan. In this study, we assembled and annotated the complete chloroplast (cp) genome sequence of A. japonica using the high-throughput sequencing approach. The cp genome of A. japonica is 153,208 bp in length with the overall GC content of 37.7%, including two inverted repeat (IR) regions of 25,147 bp, which was separated by a large single-copy (LSC) region of 84,255 bp and a small single-copy (SSC) region of 18,659 bp. 113 unique genes were annotated in the genome, including 80 protein-coding genes, 29 represented tRNA genes, and four denoted rRNA genes. A maximum-likelihood phylogenetic analysis with 38 complete cp sequences showed that Atractylodes formed a monophyletic clade, and A. japonica and A. koreana formed a subclade in Atractylodes. This study provides the chloroplast genome structure features and phylogenetic relationship of A. japonica.

Precise Species Detection in Traditional Herbal Patent Medicine, Qingguo Wan, Using Shotgun Metabarcoding.

As one of the high-incidence diseases in the world, pharyngitis seriously affects the lives of those with the condition. Qingguo Wan is a herbal medicine used for treating pharyngitis, and its quality evaluation is currently only accomplished via traditional identification. However, precise identification becomes challenging with fake products on the market or fungal contamination during the production process. This study used the Illumina NovaSeq platform for targeting the ITS2, psbA-trnH, matK, and rbcL sequences to survey the species composition of lab-made and commercial samples. The results showed that a total of 34.56 Gb of raw data that was obtained represented more than 0.23 billion reads. After assembly, annotation, and operational taxonomic unit clustering, 103, 12, 10, and 12 OTUs were obtained, which belonged to the ITS2, psbA-trnH, matK, and rbcL sequences of the mock lab-made and commercial samples. The analytical results indicated that the sequences of all the prescription ingredients were successfully obtained in the two lab-made samples. The positive control medicinal Panax quinquefolius L. sequence was obtained in HSZY175, while Scutellaria baicalensis Georgi, Lonicera japonica Thunb. Menispermum dauricum DC. and Paeonia lactiflora Pall. were detected in the three commercial samples. The detection results of the other four herbs in different fragments were not all the same. In addition, a total of 28 fungi OTUs, representing 19 families and 20 genera, were obtained from both the commercial and mock lab-made samples. Aspergillus, Cladosporium, and Penicillium dominated among the 20 genera. This study demonstrated that the shotgun metabarcoding method is a powerful tool for the molecular identification of the biological ingredients in Qingguo Wan. It can be used to effectively supplement traditional methods while providing a new technique for the quality evaluation of Qingguo Wan.

Soluplus-Mediated Diosgenin Amorphous Solid Dispersion with High Solubility and High Stability: Development, Characterization and Oral Bioavailability.

BACKGROUND AND PURPOSE: The traditional Chinese medicine, diosgenin (Dio), has attracted increasing attention because it possesses various therapeutic effects, including anti-tumor, anti-infective and anti-allergic properties. However, the commercial application of Dio is limited by its extremely low aqueous solubility and inferior bioavailability in vivo. Soluplus, a novel excipient, has great solubilization and capacity of crystallization inhibition. The purpose of this study was to prepare Soluplus-mediated Dio amorphous solid dispersions (ASDs) to improve its solubility, bioavailability and stability. METHODS: The crystallization inhibition studies were firstly carried out to select excipients using a solvent shift method. According to solubility and dissolution results, the preparation methods and the ratios of drug to excipient were further optimized. The interaction between Dio and Soluplus was characterized by differential scanning calorimetry (DSC), fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (PXRD) and molecular docking. The pharmacokinetic study was conducted to explore the potential of Dio ASDs for oral administration. Furthermore, the long-term stability of Dio ASDs was also investigated. RESULTS: Soluplus was preliminarily selected from various excipients because of its potential to improve solubility and stability. The optimized ASDs significantly improved the aqueous solubility of Dio due to its amorphization and the molecular interactions between Dio and Soluplus, as evidenced by dissolution test in vitro, DSC, FT-IR spectroscopy, SEM, PXRD and molecular docking technique. Furthermore, pharmacokinetic studies in rats revealed that the bioavailability of Dio from ASDs was improved about 5 times. In addition, Dio ASDs were stable when stored at 40°C and 75% humidity for 6 months. CONCLUSION: These results indicated that Dio ASDs, with its high solubility, high bioavailability and high stability, would open a promising way in pharmaceutical applications.

Design and evaluation of rhubarb total free anthraquinones oral colon-specific drug delivery granules to improve the purgative effect

Rhubarb is commonly used as a cathartic in Asian countries. However, researchers have devotedextensive concerns to the quality control and safety of rhubarb and traditional Chinese preparations composed of rhubarb due to the instable purgative effect and potential nephrotoxicity of anthraquinones. In this study, we aimed to prepare rhubarb total free anthraquinones (RTFA) oral colon-specific drug delivery granules (RTFA-OCDD-GN) to delivery anthraquinones to colon to produce purgative effect. RTFA-OCDD-GN were prepared using chitosan and Eudragit S100 through a double-layer coating process and the formulation was optimized. Continuous release studies were performed in a simulated gastric fluid (pH 1.2), followed by a small-intestinal fluid (pH 6.8) and a colonic fluid (pH 7.4, containing rat cecal contents). The purgative effect test was performed in rats. The dissolution profile of RTFA-OCDD-GN showed that the accumulative dissolution rate of RTFA was about 83.0% in the simulated colonic fluid containing rat cecal contents and only about 9.0% in the simulated gastrointestinal fluids. And the RTFA-OCDD-GN could produce the comparative purgative activity as rhubarb, suggesting it could deliver the free AQs to the colon. The RTFA-OCDD-GN was a useful media to enhance the purgative activity of free anthraquinones after administered orally.

Oral colon-specific drug delivery system reduces the nephrotoxicity of rhubarb anthraquinones when they produce purgative efficacy.

Rhubarb is commonly used to treat constipation in China and anthraquinones (AQs) are the active components present in rhubarb. However, an increasing number of studies have reported that AQs induce nephrotoxicity. In the present study, rhubarb total free anthraquinones (RTFA) oral colon-specific drug delivery granules (RTFA-OCDD-GN) were prepared to determine whether RTFA-OCDD-GN could reduce the nephrotoxicity that occurs when AQs produce purgative efficacy. RTFA-OCDD-GN were prepared using pH-enzyme double-layer coating technology and the cumulative release rate of RTFA in RTFA-OCDD-GN was assessed. The first black stool time, the number and state of feces over 8 h were observed to measure the purgative efficacy. In the nephrotoxicity test, biochemical and histopathological examinations were performed following 20 and 40 days administration, and 20 days convalescence. The cumulative release rate of RTFA in RTFA-OCDD-GN was >80% in simulated colonic fluid. RTFA-OCDD-GN produced considerable purgative efficacy compared with rhubarb medical material samples (RMMS). Following 40 days RMMS administration, blood urea nitrogen, creatinine and urine ß2-microglobulin levels in the high-dosage group were significantly increased compared with the control and RTFA-OCDD-GN groups (P<0.05). All specimens from the high-dosage RMMS group exhibited swelling/degeneration of renal proximal convoluted tubule epithelial cells. No difference in pathological conditions and biochemical indicators was detected between the RTFA-OCDD-GN groups and the control group. The nephrotoxicity of AQs was significantly reduced following RTFA-OCDD-GN administration, which produced considerable purgative efficacy compared with RMMS.