PAR-CLIP Assay in Ferroptosis.

Ferroptosis is a regulatory cell death process that is accompanied by large amounts of iron ion accumulation and lipid peroxidation. Photoactivated ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) is a method used to identify the binding sites of RNA-binding proteins (RBPs) on target RNAs with high resolution at the nucleotide level. By inserting photosensitive ribonucleoside analogs into new RNA transcripts of living cells, characteristic mutations can be generated during reverse transcription and be used to accurately locate the crosslinking position of RNAs and RBPs. The use of PAR-CLIP to detect interactions and determine precise crosslinking sites between RNAs and RBPs, or to search for RNAs upstream or downstream of ferroptosis pathways genes through known proteins, can help to clarify and verify the occurrence and regulation mechanisms of the various signaling pathways of ferroptosis. Furthermore, it may reveal new targets for ferroptosis detection and improve the treatment efficiency of ferroptosis-related diseases such as cancer and neurodegenerative diseases. Here, we introduce a specific PAR-CLIP protocol for monitoring the ferroptosis process.

A Discovery-Based Metabolomic Approach Using UPLC-Q-TOF-MS/MS Reveals Potential Antimalarial Compounds Present in Artemisia annua L.

In 1972, Nobel laureate Youyou Tu's research team conducted clinical trials on the dried material of Artemisia annua L. from Beijing extracted by ether and then treated with alkali (called "ether neutral dry"), which showed that artemisinin was not the only antimalarial component contained. The biosynthesis of sesquiterpenoids in A. annua has increased exponentially after unremitting cultivation efforts, and the plant resources are now quite different from those in the 1970s. In consideration of emerging artemisinin resistance, it is of great theoretical and practical value to further study the antimalarial activity of A. annua and explore its causes. The purpose of this study is to clarify scientific questions, such as "What ingredients are synergistic with artemisinin in A. annua?", and "Are there non-artemisinin antimalarial ingredients in A. annua?". In this paper, Beijing wild A. annua was used as a control and two representative cultivation species of A. annua were selected to evaluate the antimalarial activity of the herbal medicine. The antimalarial activity of different extracts on mice was studied using the Peters' four-day suppressive test. UPLC-Q-TOF-MS was used to obtain mass spectrum data for all samples, and a UNIFI platform was used for identification. A multivariate statistical method was used to screen the different compounds with positive correlations. The antimalarial activity of different components from the ether extract and alkali treatments was determined and antimalarial components other than artemisinin were obtained. A total of 24 flavonoids, 68 sesquiterpenoids and 21 other compounds were identified. Compounds associated with differential antimalarial activity were identified. The material basis for the antimalarial activity of A. annua was clarified. The antimalarial components of A. annua include two categories: first, artemisinin and non-artemisinin antimalarial active components, of which the non-artemisinin antimalarial active components may include 5α-hydroperoxy-eudesma-4(15),11-diene; second, several antimalarial synergistic ingredients in A. annua, including arteanniun B, arteanniun B analogues and polymethoxy flavonoids.

Casticin and chrysosplenol D from Artemisia annua L. induce apoptosis by inhibiting topoisomerase IIα in human non-small-cell lung cancer cells.

BACKGROUND: Artemisia annua L. (A. annua) and its active components exhibit antitumour effects in many cancer cells. However, the biological processes and mechanisms involved are not well understood, especially for the treatment of non-small-cell lung cancer (NSCLC). PURPOSE: This study aimed to comprehensively explore the biological processes of A. annua and its active components in NSCLC cells and to identify the mechanism by which these compounds induce apoptosis. STUDY DESIGNS/METHODS: Cell viability and flow cytometry assays were used to evaluate the cytotoxicity of A. annua active components casticin (CAS) and chrysosplenol D (CHD) in A. annua in NSCLC cells. After treatment with CAS and CHD, A549 cells were subjected to RNA sequencing (RNA-seq) analysis, differentially expressed genes (DEGs) were screened and subjected to functional enrichment analysis (KEGG and GO analysis) as well as protein interaction network analysis. The key targets associated with apoptosis induction in A549 cells were screened by Cytoscape, and the screened DEGs were validated by qRT-PCR. Immunoblotting, immunofluorescence, and molecular docking assays were used to determine whether CAS and/or CHD could induce apoptosis in NSCLC cells by inducing DNA damage through down-regulation of topoisomerase IIα (topo IIα) expression. The same experiments were verified again in the H1299 lung cancer cell line. RESULTS: CAS and CHD inhibited NSCLC cells proliferation in a time- and dose-dependent manner, and significantly induced apoptosis. A total of 115 co-upregulated DEGs and 277 co-downregulated DEGs were identified in A549 cells following treatment with CAS and CHD. Comprehensive and systematic data about biological processes and mechanisms were obtained. DNA damage pathways and topo IIα targets were screened to study the apoptosis effects of CAS and CHD on NSCLC cells. CAS and CHD may be able to induce DNA damage by binding to topo IIα-DNA and reducing topo IIα activity. CONCLUSION: This study suggested that CAS and CHD may reduce topo IIα activity by binding to topo IIα-DNA, affecting the replication of DNA, triggering DNA damage, and inducing apoptosis. It described a novel mechanism associated with topo IIα inhibition to reveal a novel role for CAS and CHD in A. annua as potential anticancer agents and/or adjuvants in NSCLC cells.

Affinity ultrafiltration and UPLC-HR-Orbitrap-MS based screening of thrombin-targeted small molecules with anticoagulation activity from Poecilobdella manillensis.

This study aims to screen potential anticoagulant components from leeches, a representative animal-sourced traditional Chinese medicine using thrombin (THR)-targeted ultrafiltration combined with ultrahigh performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). As a result, five small molecules in leech extract were discovered to interact with THR for the first time. Among them, two new compounds were isolated and their structures were identified by IR, HR-MS and NMR data. Furthermore, their THR inhibitory activity was confirmed with IC50 values of 4.74 and 8.31 µM, respectively. In addition, molecular docking analysis showed that the active (catalytic) site of THR might be the possible binding site of the two hits. Finally, reverse screening analysis indicated that LTA4-H, ACE and ALOX5AP were potential anticoagulant targets of the two new compounds. This study will broaden our understanding of the medicinal substance basis in leeches and further contribute to the discovery and development of clinical anticoagulant drugs from leeches.

Heparin-decorated nanostructured lipid carriers of artemether-protoporphyrin IX-transferrin combination for therapy of malaria.

Heme is a prosthetic group of hemoglobin comprising protoporphyrin IX (PPIX) with Fe2+. Studies have shown that modulating heme synthesis pathway in Plasmodium could greatly affect the action mechanism and antimalarial effect of artemisinin and its derivatives. Herein, an intraerythrocytic parasite targeted nanostructured lipid carrier (NLC) was developed for potentiation of artemether (ARM) by combination with PPIX and iron-loaded transferrin (holo-Tf). Firstly, ARM and PPIX were co-loaded into NLCs with high entrapment efficiency. Then, a targeting ligand heparin (HP) was electrostatically adsorbed onto the periphery of NLCs, followed by conjugation with holo-Tf to receive the final formulation Tf-HP-NLC/ARM/PPIX. Tf-HP-NLC/ARM/PPIX exhibited nanoscale particle size (~188 nm) and was relatively stable in simulated gastrointestinal fluids and rat plasma. A sustained drug release characteristic was observed in PBS (pH 7.4). In vitro targeting assay confirmed that Tf-HP-NLC/ARM/PPIX could be specifically and efficiently internalized into intraerythrocytic parasites via HP receptor-meditated endocytosis. Furthermore, due to enhanced intraparasitic accumulation and activated mechanism of ARM, the combinational delivery system Tf-HP-NLC/ARM/PPIX showed increased inhibitory activity against Plasmodium falciparum in culture and enhanced antimalarial effect in Plasmodium berghei-infected murine model, suggesting a promising strategy for development of new therapy based on action mechanism of ARM.

[Research advances of biosynthesis, in vivo analysis and pharmacokinetics of chemical constituents in Artemisiae Annuae Herba].

Artemisiae Annuae Herba is a traditional Chinese medicine for clearing deficiency and heat. It is the only natural source of artemisinin, which is a specific antimalarial drug, and has been widely concerned all over the world. In addition to artemisinin, Artemisiae Annuae Herba also contains many sesquiterpenes, coumarins, flavonoids, volatile oils, polysaccharides and other chemical components, which show antipyretic, anti-inflammatory, antiviral microorganisms, anti-asthma, anti-oxidation, anti-tumor and other pharmacological activities. In addition to their own pharmacological activities, some components could enhance the antimalarial activity of artemisinin through different mechanisms at absorption and metabolism in vivo. In order to understand the pharmacokinetic characte-ristics of the chemical constituents contained in Artemisiae Annuae Herba and provide reference for the full development and clinical utilization of Artemisiae Annuae Herba resources in China, this present paper systematically collated the modern research literatures, and summarized the biosynthesis, in vivo analysis and pharmacokinetics of the chemical constituents in Artemisiae Annuae Herba.

Oral Bioavailability Comparison of Artemisinin, Deoxyartemisinin, and 10-Deoxoartemisinin Based on Computer Simulations and Pharmacokinetics in Rats.

Deoxyartemisinin, a compound separated from Artemisinin annua L., shows anti-inflammatory and antiulcer activities. 10-Deoxoartemisinin is a novel compound with a strong antimalarial effect derivatized from artemisinin. Compared to the famous antimalarial natural compound artemisinin, deoxyartemisinin lacks the peroxide bridge structure, while 10-deoxoartemisinin remains this special peroxide bridge group but loses the 10-position keto group. To clarify their pharmacological differences, the absorption, distribution, metabolism, excretion (ADME) properties of artemisinin, deoxyartemisinin, and 10-deoxoartemisinin were first predicted using QikProp software. Also, their pharmacokinetic behaviors in rats were further evaluated by a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method after oral and intravenous administration of each compound, in which deoxyartemisinin and 10-deoxoartemisinin were first evaluated for their pharmacokinetics. All parameters about ADME properties calculated by software met the criteria and the ADME performance order was 10-deoxoartemisinin > deoxyartemisinin > artemisinin. The oral bioavailability of artemisinin was calculated to be 12.2 ± 0.832%, which was about 7 times higher than that of deoxyartemisinin (1.60 ± 0.317%). For 10-deoxoartemisinin, its bioavailability (26.1 ± 7.04%) was superior to artemisinin at a degree of more than twice. Considering their chemical structures, losing the peroxide bridge might decrease the absorption rate of deoxyartemisinin in the gastrointestinal tract, while retaining the peroxide bridge but losing the 10-position ketone might improve the bioavailability of 10-deoxoartemisinin.

Phytochemical analysis and geographic assessment of flavonoids, coumarins and sesquiterpenes in Artemisia annua L. based on HPLC-DAD quantification and LC-ESI-QTOF-MS/MS confirmation.

To evaluate the quality of Artemisia annua L., an accurate HPLC-DAD method has been developed, validated and applied to the simultaneous quantification of five flavonoids, two coumarins and four sesquiterpenes. An LC-ESI-QTOF-MS/MS confirmation method has been utilized to avoid false-positive results. Principal component analysis and hierarchical cluster analysis results indicated that their contents had obvious regional characteristics. Samples with high contents of artemisinin (8.24 ± 2.92 mg/g) and isorhamnetin (0.28 ± 0.25 mg/g) are mainly distributed south of the Yangtze River, and samples with high contents of scopolin (0.46 ± 0.22 mg/g), scopoletin (1.05 ± 0.17 mg/g), chrysosplenol D (0.64 ± 0.14 mg/g), casticin (1.07 ± 0.23 mg/g), arteannuin B (0.69 ± 0.18 mg/g) and artemisinic acid (3.02 ± 1.00 mg/g) are mainly distributed in eastern and northern China. Geographic content differences of the components in A. annua indicate the potential differences in the health-promoting effects of its clinical application.

Simultaneous Quantification of Five Sesquiterpene Components after Ultrasound Extraction in Artemisia annua L. by an Accurate and Rapid UPLC⁻PDA Assay.

Objective: To develop an accurate and rapid ultra-performance liquid chromatography (UPLC) coupled with a photodiode array (PDA) method for the simultaneous determination of artemisinin (Art), arteannuin B (Art B), arteannuin C (Art C), dihydroartemisinic acid (DHAA) and artemisinic acid (AA) in Artemisia annua L. Methodology: Chromatography separation was performed on an ACQUITY UPLC BEH C18 Column with isocratic elution; the mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B) (A:B = 40:60, v/v). Data were recorded at an ultraviolet (UV) wavelength of 191 nm for Art, Art C, DHAA and AA, and 206 nm for Art B. Results: The calibration curves of the five sesquiterpene components were all linear with correlation coefficients more than 0.9990. The linear ranges were 31.44-1572 µg/mL, 25.48-1274 µg/mL, 40.56-2028 µg/mL, 31.44-1572 µg/mL and 26.88-1396 µg/mL for Art, Art B, Art C, DHAA and AA, respectively. The precision ranged from 0.08% to 2.88%, the stability was from 0.96% to 1.66%, and the repeatability was all within 2.42% and had a mean extraction recovery of 96.5% to 100.6%. Conclusion: The established UPLC-PDA method would be valuable for improving the quantitative analysis of sesquiterpene components in Artemisia annua L.

Safety evaluation of Semen Sojae Preparatum based on simultaneous LC-ESI-MS/MS quantification of aflatoxin B1 , B2 , G1 , G2 and M1.

Semen Sojae Preparatum (SSP) is one of the most widely used traditional Chinese medicines, and is also a functional food. However, contamination with aflatoxins may occur in the fermentation process. To evaluate its safety, an accurate and rapid LC-ESI-MS/MS analytical method was developed and validated for the simultaneous determination of AFB1 , AFB2 , AFG1 , AFG2 and AFM1 in SSP. After a simple ultrasonic extraction of SSP samples, chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (2.1 × 50 mm, 3.5 µm) with a flow rate of 0.50 mL/min. The gradient elution program was performed using a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. Detection of five aflatoxins was based on triple quadrupole mass spectrometry using a multiple reaction monitoring mode with an electrospray ionization source. SSP is likely to be contaminated by aflatoxins in the processes of fermentation, storage, transportation and usage, and it is necessary to strictly monitor it. Artemisia annua L. and Morus alba L. may inhibit the production and growth of AFB1 - and AFB2 -producing fungi, which has a certain detoxification effect on contamination with aflatoxins in SSP.

[Construction of artesunate nanoparticles modified by hyaluronic acid and cell-penetrating peptides and its inhibitory effect on cancer cells in vitro].

Hyaluronic acid (HA) and cell-penetrating peptide (CPP) R6H4-SA modified artesunate nanostructured lipid carrier (HA-R6H4-NLC/ART) for anti-tumor therapy was prepared. The physicochemical properties and in vitro drug release of HA-R6H4-NLC/ART were evaluated, and the uptake and cytotoxicity of liver cancer HepG2 cells were studied. The results showed that HA-R6H4-NLC/ART was spherical like in appearance, and the average particle size was about 160 nm. In vitro release experiments showed that the drug delivery system had sustained release characteristics. Cell results showed that, in slightly acidic environment, pH sensitive CPP R6H4-SA mediated cellular uptake of nanoparticles was significantly higher than that of non-sensitive peptide R8-SA. Meanwhile, HA-R6H4-NLC/ART had a targeting effect on HepG2 cells, and the HA receptor saturation experiment showed that the endocytosis of HA-R6H4-NLC/ART was mediated by the HA receptor on the cell surface. As compared with the unmodified or R6H4-SA single modified group, HA and R6H4-SA co-modified HA-R6H4-NLC/ART significantly improved the cell uptake and had a stronger anti-tumor effect under the conditions of the slightly acid environment and hyaluronidase degradation. The above results showed that hyaluronic acid and CPP R6H4-SA co-modified artesunate nanostructured lipid carrier, which can effectively identify and penetrate the tumor cell membrane into the cell, is a potentially efficient targeting delivery system for anti-tumor drugs.

Simultaneous high-performance liquid chromatography with diode array detection and time-of-flight mass spectrometric confirmation of the ten bioactive compounds in Semen Sojae Preparatum.

Semen Sojae Preparatum is one of the most widely used traditional Chinese medicines. A reliable and accurate high-performance liquid chromatography with diode array detection method has been developed and validated for the quantitative determination of the ten bioactive compounds contained in Semen Sojae Preparatum. The samples were first extracted by pressurized liquid extraction using 80% ethanol at 100°C for 15 min and three static extraction cycles. Chromatographic separation was conducted on a C18 column using a mobile phase consisting of water and acetonitrile under gradient elution, and the detection wavelength was set at 210 nm. The samples were further analyzed on a high-performance liquid chromatography with time-of-flight mass spectrometry system to confirm the determination results. All the ten analytes were well separated, and the calibration curves showed good linearity. The intra- and interday precisions were evaluated in terms of relative standard deviation values within the ranges of 0.20-1.43% and 0.40-4.78%, respectively. The recoveries for the ten analytes were all in the ranges of 96.2-104.3%, with relative standard deviation values < 3.85%. The established high-performance liquid chromatography method could serve as a reliable and accurate method for the quality evaluation of Semen Sojae Preparatum from different origins.

[History of processing of Sojae Semen Praeparatum].

Sojae Semen Praeparatum (SSP) is commonly used as a type of dietetic Chinese herb. By collecting and analyzing ancient and recent literatures, a textual criticism was conducted on the historical evolution of the processing of SSP. Fermented soybean was recorded in Shijing, and relevant rational processing was described in Qimin Yaoshu. In the early time, fermented soybean included the type of "salty" and "light". After the Ming Dynasty, "light" fermented soybean or SSP was recognized as a better medicinal matter than salty fermented soybean, and the fermentation processing was recorded more clearly. In modern time, many characteristic methods for processing SSP have been developed. Today, the processing of SSP is mainly based on the Chinese Pharmacopoeia, which records soybean as a main ingredient and Artemisiae Annuae Herba, Mori Folium as excipients.

Combination of artemisinin-based natural compounds from Artemisia annua L. for the treatment of malaria: Pharmacodynamic and pharmacokinetic studies.

Currently, the most effective antimalarial is artemisinin, which is extracted from the leaves of medicinal herb Artemisia annua L. (A. annua). Previous studies showed that the complex chemical matrix of A. annua could enhance both the bioavailability and efficacy of artemisinin. The present study aims to evaluate the efficacy and pharmacokinetic properties of a combination therapy based on artemisinin and 3 components from A. annua with high content (arteannuin B, arteannuic acid, and scopoletin). In vivo antimalarial activity was assessed following a 4-day treatment in murine malaria models (Plasmodium yoelii and Plasmodium berghei). Results showed that a much sharper reduction in parasitemia (~93%) was found in combination therapy compared with pure artemisinin (~31%), indicating pharmacodynamic synergism occurring between artemisinin and arteannuin B, arteannuic acid, and scopoletin. Multiple-dose pharmacokinetics further demonstrated that combination therapy results in increased area under the curve (AUC0→∞ ), Cmax , and t1/2 by 3.78-, 3.47-, and 1.13-fold in healthy mice, respectively, and by 2.62-, 1.82-, and 1.22-fold in P. yoelii-infected mice, respectively. The calculated oral clearance of combination therapy in healthy and P. yoelii-infected mice was also reduced. These findings imply that specific components in A. annua might offer a possibility to develop new artemisinin-based natural combination therapy for malaria treatment.

Free paclitaxel-loaded E-selectin binding peptide modified micelle self-assembled from hyaluronic acid-paclitaxel conjugate inhibit breast cancer metastasis in a murine model.

The present work seeks to construct a nanovehicle for the efficient suppression of breast cancer metastasis through targeting E-selectin on tumor vascular endothelial cells and hyaluronic acid-receptor on tumor cells. Herein, a new ligand-PEG-lipid conjugate, E-selectin binding peptide-polyethene glycol-1-octadecylamine (Esbp-PEG-OA), was used as the targeting molecule of micelle self-assembled form hyaluronic acid-paclitaxel (HA-PTX) conjugate. When loaded with free PTX, the micelles (Esbp-HA-PTX/PTX) exhibited nanoscale particle size with high drug-loading capacity (up to 31.5%). In vitro release study showed that the conjugated and entrapped PTX released simultaneously. Cellular uptake of micelles confirmed that Esbp-HA-PTX micelles could be specifically and efficiently internalized into E-selectin expressing human umbilical vein endothelial cells (HUVEC) and 4T1 breast cancer cells via receptor-meditated endocytosis. In vitro cytotoxicity assay further revealed that Esbp-HA-PTX/PTX micelles significantly improved the selectivity of PTX for killing the two cell types compared with PTX solution formulation. More importantly, Esbp-HA-PTX micelles raised the accumulation of payload in tumor through targeting two cell types in the tumor microenvironment simultaneously, resulting in marked in vivo inhibition of tumor growth, intratumoral microvessel density and metastasis, and decreased systemic toxicity over solution formulation. Overall, Esbp-HA-PTX/PTX micelle is promising in therapy of breast cancer metastasis.

Two new guaiane-type sesquiterpenoids from Valeriana hardwickii and their cytotoxicity.

Two new guaiane-type sesquiterpenoids, named 4α,5α-epoxy-8ß-hydroxy-1α-hydro-α-guaiene (1) and 4α,5α-epoxy-1-hydroxy-α-guaiene (2), were isolated from the whole plants of Valeriana hardwickii. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1 and 2 showed weak cytotoxicity against the lung adenocarcinoma (A549) and hepatoma (Bel7402) cell lines with IC50 values of 9.2 and 8.5 µM, respectively.

Artemisinin and Its Derivatives as a Repurposing Anticancer Agent: What Else Do We Need to Do?

Preclinical investigation and clinical experience have provided evidence on the potential anticancer effect of artemisinin and its derivatives (ARTs) in the recent two decades. The major mechanisms of action of ARTs may be due to toxic-free radicals generated by an endoperoxide moiety, cell cycle arrest, induction of apoptosis, and inhibition of tumor angiogenesis. It is very promising that ARTs are expected to be a new class of antitumor drugs of wide spectrum due to their detailed information regarding efficacy and safety. For developing repurposed drugs, many other characteristics of ARTs should be studied, including through further investigations on possible new pathways of anticancer effects, exploration on efficient and specific drug delivery systems-especially crossing biological barriers, and obtaining sufficient data in clinical trials. The aim of this review is to highlight these achievements and propose the potential strategies to develop ARTs as a new class of cancer therapeutic agents.

Effects of arteannuin B, arteannuic acid and scopoletin on pharmacokinetics of artemisinin in mice.

OBJECTIVE: To explore the effects of arteannuin B, arteannuic acid and scopoletin on the pharmacokinetics of artemisinin in mice. METHODS: Artemisinin and a combination of artemisinin, arteannuin B, arteannuic acid and scopoletin were administered together to mice via oral administration. Blood samples were collected at different time intervals and pretreated by liquid-liquid extraction. The contents of four compounds in mouse plasma were determined by a validated HPLC-MS/MS method. RESULTS: Compared to single artemisinin group, the Cmax values from the combination group rose from 947 ng/mL to 1254 ng/mL. AUC(0-t) (2371 h ng/mL) was significantly higher than that from single artemisinin group (747 h ng/mL). The peak time lag and the CL values reduced at a proportion of 66%. CONCLUTIONS: Arteannuin B, arteannuic acid and scopoletin can markedly affect the pharmacokinetics of artemisinin.

Liver-targeting self-assembled hyaluronic acid-glycyrrhetinic acid micelles enhance hepato-protective effect of silybin after oral administration.

In order to enhance oral bioavailability and liver targeting delivery of silybin, two amphiphilic hyaluronic acid derivatives, hyaluronic acid-deoxycholic acid (HA-adh-DOCA) and hyaluronic acid-glycyrrhetinic acid (HA-adh-GA) conjugates, were designed and synthesized. Silybin was successfully loaded in HA-adh-DOCA and HA-adh-GA micelles with high drug-loading capacities (20.3% ± 0.5% and 20.6% ± 0.6%, respectively). The silybin-loaded micelles were spherical in shape with the average size around 130 nm. In vitro release study showed that two silybin-loaded micelles displayed similar steady continued-release pattern in simulated gastrointestinal fluids and PBS. Single-pass intestinal perfusion studies indicated that silybin-loaded micelles were absorbed in the whole intestine and transported via a passive diffusion mechanism. Compared with suspension formulation, silybin-loaded HA-adh-DOCA and HA-adh-GA micelles achieved significantly higher AUC and Cmax level. Moreover, liver targeting drug delivery of micelles was confirmed by in vivo imaging analysis. In comparison between the two micellar formulations, HA-adh-GA micelles possessed higher targeting capacity than HA-adh-DOCA micelles, owing to the active hepatic targeting properties of glycyrrhetinic acid. In the treatment of acute liver injury induced by CCl4, silybin-loaded HA-adh-GA micelles displayed better effects over suspension control and silybin-loaded HA-adh-DOCA micelles. Overall, pharmaceutical and pharmacological indicators suggested that the HA-adh-GA conjugates can be successfully utilized for liver targeting of orally administered therapeutics.

Determination of astragaloside III in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study.

Astragaloside III (AST III), a naturally occurring saponin compound isolated from Radix Astragali, has been demonstrated to have anti-gastric ulcer, immunomodulatory and antitumor effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method has been developed and validated for the quantification of astragaloside III in rat plasma. Samples were pretreated using a simple protein precipitation with methanol-acetonitrile (50:50, v/v) and the chromatographic separation was performed on a C18 column by a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Astragaloside III and the internal standard (buspirone) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range of 5.00-5000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that astragaloside III spiked into rat plasma was stable for 24 h at 20°C temperature, for up to 30 days at -80°C, and during three freeze-thaw cycles. The method was successfully used to investigate the pharmacokinetic profile of AST III after oral (10 mg/kg) and intravenous (1.0 mg/kg) administration in rats. The oral absolute bioavailability of AST III was calculated to be 4.15 ± 0.67% with an elimination half-life value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo.