Sibao Chen

Comparative Physicochemical Characterization of Phospholipids Complex of Puerarin Formulated by Conventional and Supercritical Methods

PurposeThe aim of this work was to compare the physicochemical characteristics of the phospholipids complex of puerarin (Pur) prepared by traditional methods (solvent evaporation, freeze-drying and micronization) and a supercritical fluid (SCF) technology. The physicochemical properties of the pure drug and the corresponding products prepared by two different SCF methods were also compared.MethodsSolid-state characterization of particles included differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), solubility, dissolution rate and scanning electron microscopy (SEM) examinations. Besides puerarin phospholipids complex (PPC) by four different methods, the solid-state properties of unprocessed, gas antisolvent (GAS) crystallized and solution enhanced dispersion by supercritical fluid (SEDS) precipitated puerarin samples were also compared. Crystallinity was assessed using DSC and XRPD. Drug-phospholipids interactions were characterized using Fourier transform infrared spectroscopy (FTIR). SEM was used to determine any morphological changes. Pharmaceutical performance was assessed in dissolution rate and solubility tests.ResultThe results of the physical characterization attested a substantial correspondence of the solid state of the drug before and after treatment with GAS technique, whereas a pronounced change in size and morphology of the drug crystals was noticed. The GAS-processed puerarin exhibited a better crystal shape confirmed by DSC, XRPD and IR. Polymorphic change of puerarin during SEDS coupled with the dramatic reduction of the dimensions determined a remarkable enhancement of its solubility and in vitro dissolution rate. Phospholipids complex prepared using supercritical fluid technology showed similar properties of physical state, thermal stability and molecular interaction with phospholipids (PC) to those of corresponding systems prepared by other three conventional methods namely solvent evaporation, freeze-drying and micronization as proved by XRPD, DSC, and FTIR. The best dissolution rate was obtained by SEDS-prepared complex, while the highest solubility was obtained for solvent evaporation method.ConclusionSupercritical fluid technology for the preparation of puerarin and its phospholipids complex has been proven to have significant advantages over the solvent evaporation technique and other conventional methods.

Proteomic identification of differentially expressed proteins in curcumin-treated MCF-7 cells

Curcumin (CM), a well-known dietary pigment derived from Curcuma longa L., possess anticancer activities against a variety of tumors including human breast carcinoma. In combination with docetaxel, CM has been used in breast cancer management in the clinic. In order to explore the possible mechanism of anticancer activity of CM, in the present study, we aimed to identify proteins involved in the anticancer activity of CM in human breast cancer cell line MCF-7 using the two-dimensional electrophoresis (2-DE)-based proteomic analysis. MCF-7 cells were cultured at 37°C in an atmosphere of 5.0% CO(2). All the following experiments were repeated three times. Cell viability assay showed that after a 48-h incubation CM dose-dependently inhibited cell growth with an IC(50) value of 47.42 μM. Treatment of CM at 47.42 μM for 48 h induced apoptosis as determined by nuclear morphologic changes of Hoechst stained cells and flow cytometric analysis of Annexin V-FITC/PI stained cells. Proteomic analysis identified 12 differentially expressed proteins which contributed to multiple functional activities such as DNA transcription, mRNA splicing and translation, amino acid synthesis, protein synthesis, folding and degradation, lipid metabolism, glycolysis, and cell motility. Among them 7 proteins were up-regulated and 5 down-regulated. The up-regulated ones were verified by quantitative real-time PCR. The down-regulated proteins, TDP-43, SF2/ASF and eIF3i, as well as up-regulated ones, 3-PGDH, ERP29, and platelet-activating factor acetylhydrolase IB subunit beta positively contribute to the anticancer activity of CM in MCF-7 cells. These molecules are implicated in the bioactivities of CM for the first time. The findings of this study would shed new insights for systematically understanding the mechanisms of CM in breast cancer intervention.

Process parameters and morphology in puerarin, phospholipids and their complex microparticles generation by supercritical antisolvent precipitation

The aim of the study was to develop and evaluate a new method for the production of puerarin phospholipids complex (PPC) microparticles. The advanced particle formation method, solution enhanced dispersion by supercritical fluids (SEDS), was used for the preparation of puerarin (Pur), phospholipids (PC) and their complex particles for the first time. Evaluation of the processing variables on PPC particle characteristics was also conducted. The processing variables included temperature, pressure, solution concentration, the flow rate of supercritical carbon dioxide (SC-CO2) and the relative flow rate of drug solution to CO2. The morphology, particle size and size distribution of the particles were determined. Meanwhile Pur and phospholipids were separately prepared by gas antisolvent precipitation (GAS) method and solid characterization of particles by the two supercritical methods was also compared. Pur formed by GAS was more orderly, purer crystal, whereas amorphous Pur particles between 0.5 and 1microm were formed by SEDS. The complex was successfully obtained by SEDS exhibiting amorphous, partially agglomerated spheres comprised of particles sized only about 1microm. SEDS method may be useful for the processing of other pharmaceutical preparations besides phospholipids complex particles. Furthermore adopting a GAS process to recrystallize pharmaceuticals will provide a highly versatile methodology to generate new polymorphs of drugs in addition to conventional techniques.

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B(4), respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.

Simultaneous Determination of Eight Anthraquinones in Semen Cassiae by HPLC-DAD

INTRODUCTION Semen Cassiae (SC), a traditional Chinese herbal medicine for the treatment of various diseases, is known to contain active anthraquinone ingredients. However, since the content of some anthraquinones is too low, previous analytical methods only allow the quantitation of a few anthraquinones or a hydrolysis step has to be included in the sample preparation. A rapid and accurate method to examine the content of as many anthraquinones as possible in SC would be desirable. OBJECTIVE To develop a rapid, sensitive and accurate high-performance liquid chromatography-diode array detection (HPLC- DAD) method to simultaneously quantify eight major anthraquinones (obtusifolin-2-glucoside, aurantio-obtusin, aloe- emodin, rhein, obtusifolin, emodin, chrysophanol and physcion) in SC. METHODOLOGY The separation of anthraquinones was achieved on a C₁₈-column with a gradient elution using acetonitrile and 0.1% phosphoric acid. The detection wavelength was 278 nm and the analysis finished within 25 min. RESULTS The limits of detection of these compounds ranged from 0.07 to 0.15 µg/mL while the limits of quantitation ranged from 0.24 to 0.51 µg/mL. All calibration curves showed good linearities (r²  > 0.999) within the test ranges. This validated method was successfully used to analyse 22 batches of SC samples collected from various geographical locations. CONCLUSION The method was validated to be simple, rapid, accurate and reliable to simultaneously determine eight major anthraquinones in SC. Meanwhile, a more specific anthraquinone, obtusifolin, was proposed to serve as a marker for SC, replacing chrysophanol.

Anti-inflammatory activities and mechanisms of action of the petroleum ether fraction of Rosa multiflora Thunb. hips.

ETHNOPHARMACOLOGICAL RELEVANCE The hip of Rosa multiflora Thunb. has been traditionally used as a dietary supplement and a herbal remedy for the treatment of various diseases including cold, flu, inflammation, osteoarthritis, rheumatoid arthritis and chronic pain in China. AIMS OF THE STUDY To explore the anti-inflammatory ingredient of the hip of R. multiflora Thunb. and its mechanism of action. MATERIALS AND METHODS The ethanol extract of the hip of R. multiflora Thunb. was fractioned with petroleum ether, ethyl acetate and water, and each fraction was screened for anti-inflammatory activity in xylene-induced mouse ear edema model. Three more models, acetic acid-induced mouse vascular permeation, cotton pellet-induced rat granuloma, and carrageenan-induced rat hind paw edema were also employed to verify the anti-inflammatory effect of the identified fraction. To explore the mechanism of action, the activity of inducible nitric oxide synthase (iNOS) and the level of nitric oxide (NO) in sera, as well as mRNA expression level of cyclo-oxygenase-2 (COX-2) in inflammatory tissues of rats with carrageenan-induced hind paw edema were measured. GC-MS technology was applied to identify the active components in the active fraction. RESULTS AND CONCLUSIONS The petroleum ether fraction (PEF) was identified to be the active fraction in inflammation animal models (i.e., oral administration of PEF (168.48, 42.12 and 10.53 mg/kg) evoked a significantly (P<0.001) dose-dependent inhibition of the xylene-induced mice ear edema). Down-regulating COX-2 expression (P<0.001) and reducing NO production (P<0.05) through inhibiting iNOS activity (P<0.001) may be the partial mechanism of action of PEF. GC-MS analysis indicated that unsaturated fatty acids are enriched in PEF and may be responsible for the anti-inflammatory activity of PEF and this herb. The results of this study provide pharmacological and chemical basis for the application of the hip of R. multiflora Thunb. in inflammatory disorders.

New Perspectives on Chinese Herbal Medicine (Zhong-Yao) Research and Development

Synthetic chemical drugs, while being efficacious in the clinical management of many diseases, are often associated with undesirable side effects in patients. It is now clear that the need of therapeutic intervention in many clinical conditions cannot be satisfactorily met by synthetic chemical drugs. Since the research and development of new chemical drugs remain time-consuming, capital-intensive and risky, much effort has been put in the search for alternative routes for drug discovery in China. This narrative review illustrates various approaches to the research and drug discovery in Chinese herbal medicine. Although this article focuses on Chinese traditional drugs, it is also conducive to the development of other traditional remedies and innovative drug discovery.

Atractylenolide II induces G1 cell-cycle arrest and apoptosis in B16 melanoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE Atractylenolide II (AT-II) is a sesquiterpene compound isolated from the dried rhizome of Atractylodes macrocephala (Baizhu in Chinese), which is traditionally prescribed for melanoma treatment by Chinese medicine practitioners. Our previous study showed that AT-II can inhibit B16 cells proliferation. Here we investigate the mechanistic basis for the anti-proliferative activity of AT-II in B16 melanoma cells. MATERIALS AND METHODS Cell viability was examined by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein expression was determined by Western blotting. RESULTS AT-II treatment for 48 h dose-dependently inhibited cell proliferation with an IC(50) of 82.3 μM, and induced G1 phase cell cycle arrest. Moreover, treatment with 75 μM AT-II induced apoptosis. These observations were associated with the decrease of the expression of Cdk2, phosphorylated-Akt, phosphorylated-ERK and Bcl-2, the increase of the expression of phosphorylated-p38, phosphorylated-p53, p21, p27, and activation of caspases-8, -9 and -3. In addition, a chemical inhibitor of p53, PFTα, significantly decreased AT-II-mediated growth inhibition and apoptosis. CONCLUSIONS We demonstrated that the G1-arresting and apoptotic effects of AT-II in B16 cells involve p38 activation as well as ERK and Akt inactivation, and the cytotoxic/apoptotic effects of AT-II are potentially p53 dependent. These findings provided chemical and pharmacological basis for the traditional application of Baizhu in melanoma treatment.

New Perspectives on Innovative Drug Discovery: An Overview

Despite advances in technology, drug discovery is still a lengthy, expensive, difficult, and inefficient process, with a low rate of success. Today, advances in biomedical science have brought about great strides in therapeutic interventions for a wide spectrum of diseases. The advent of biochemical techniques and cutting-edge bio/chemical technologies has made available a plethora of practical approaches to drug screening and design. In 2010, the total sales of the global pharmaceutical market will reach 600 billion US dollars and expand to over 975 billion dollars by 2013. The aim of this review is to summarize available information on contemporary approaches and strategies in the discovery of novel therapeutic agents, especially from the complementary and alternative medicines, including natural products and traditional remedies such as Chinese herbal medicine.

Petroleum ether extractive of the hips of Rosa multiflora ameliorates collagen-induced arthritis in rats

ETHNOPHARMACOLOGICAL RELEVANCE The hip of Rosa multiflora Thunb. (HRM) has been traditionally used as a dietary supplement and a herbal remedy for the treatment of various diseases, including inflammation, osteoarthritis, rheumatoid arthritis and chronic pain, in China. The current study was to evaluate the therapeutic efficacy of the petroleum ether extractive of HRM (PEE) on type II collagen-induced rheumatoid arthritis (CIA) in male Wistar rats. In addition, the anti-inflammatory mechanism(s) of PEE on type II CIA was explored. MATERIALS AND METHODS Rheumatoid arthritis (RA) was induced by intradermal injection of bovine type II collagen on Day 1 and Day 8. Starting from Day 13, normal rats were treated with vehicle (serving as the control group); the CIA rats were treated with vehicle (CIA group), dexamethasone (0.25mg/kg bw per day, p.o.) (a positive control), lei-gong-teng (LGT: 10mg/kg bw per day, p.o.) (a clinically used Chinese patent medicine in RA therapy) or PEE (12, 36 or 120mg/kg bw per day, p.o.) for 28 days. RESULTS AND CONCLUSIONS PEE (120mg/kg bw per day) efficiently attenuated the severity of arthritis in the CIA rats by reducing the mean arthritis severity scores and the fore/hind paw swelling as well as reduced histological changes by decreasing the cartilage surface erosion and cartilage proteoglan depletion. PEE׳s therapeutic effect in RA may involve the inhibition of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, in serum and/or the elevation of the activities of hepatic anti-oxidative enzymes including SOD, CAT and GSH-Px. However, the detailed anti-inflammatory mechanism, the main effective components and the interaction between different ingredients in PEE are still not clear and require more studies.