Nani Wang

Pipette tip solid-phase extraction and high-performance liquid chromatography for the determination of flavonoids from Epimedii herba in rat serum and application of the technique to pharmacokinetic studies

Epimedii herba is a traditional Chinese medicine for the treatment of osteoporosis. Epimedin A, B and C and icariin are the primary effective ingredients of this medicine. In this study, a simple and low-cost method based on pipette tip solid-phase extraction, high-performance liquid chromatography separation, and diode array detection has been developed for the simultaneous analysis of four flavonoids (epimedin A, B and C and icariin) from Epimedii herba in rat serum samples. In this novel extraction configuration, the sorbents were placed between a filter (hollow fiber) and the pipette tip. Pipette tip solid-phase extraction has several advantages compared to conventional extraction methods: faster extraction time (6.0min); lower sample volume (100μL); lower solvent volume (100μL); and less solvent waste. Under the optimum extraction conditions, the method showed good linearity (0.05-10.0μgmL(-1)), acceptable intra- and inter precision (RSD<6%), low limits of quantification (0.027-0.045μgmL(-1)) and satisfactory relative recoveries (98.63-103.18%). This method was successfully applied to investigate the pharmacokinetics of the major flavonoids in Epimedii herba extract after oral administration to rats (10gkg(-1) body weight). The primary pharmacokinetic parameters for rats were determined as follows: Cmax, 0.45-4.11μgmL(-1); Tmax, 0.21-0.26h; t1/2α, 0.06-0.12h; t1/2β, 2.02-3.48h; AUC0-∞: 0.50-2.58μghmL(-1); CL, 19.53-44.72Lkg(-1)h(-1); and MRT0-∞, 2.25-3.77h. The developed method has the potential to promulgate the pharmacokinetics and provide more information for clinical applications.

Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods.

Poria cocos Wolf is a popular traditional medicinal plant that has invigorating activity. Water-soluble polysaccharides (PCPs) are its main active components. In this study, four different methods were used to extract PCPs, which include hot water extraction (PCP-H), ultrasonic-assisted extraction (PCP-U), enzyme-assisted extraction (PCP-E) and microwave-assisted extraction (PCP-M). Their chemical compositions and structure characterizations were compared. In vitro antioxidant activities were studied on the basis of DPPH radical, hydroxyl radical, reducing power and metal chelating ability. The results showed that PCPs were composed of mannose, glucose, galactose, and arabinose, and had typical IR spectra characteristics of polysaccharides. Compared with other PCPs, PCP-M had lower neutral sugar content, higher mannose content and higher uronic acid content. The molecular weight were determined as PCP-E<PCP-M<PCP-U<PCP-H. PCP-M showed the strongest reducing power and highest scavenging abilities on hydroxyl and DPPH radicals, while PCP-U exhibited the lowest antioxidant activities. Response surface methodology was used to optimize the extraction yield of PCP-M by implementing the Box-Behnken design. Under the optimized conditions, the PCP-M yield was 9.95%, which was well in close agreement with the value predicted by the model. Overall, the microwave-assisted extraction was an effective and mild method for obtaining antioxidant polysaccharides from P. cocos Wolf.

Carbon nanotube-polymer composite for effervescent pipette tip solid phase microextraction of alkaloids and flavonoids from Epimedii herba in biological samples

An effervescent pipette tip solid phase microextraction based on carbon nanotube-polymer composite microspheres was developed for the simultaneous extraction and determination of four alkaloids (magnoflorine, epiberberine, palmatine, and jatrorrhizine) and four flavonoids (epimedin A/B/C and icariin) from Epimedii herba in biological samples. In this work, the polymeric sorbent was prepared by incorporating carbon nanotube in sulfonated polystyrene-divinylbenzene microspheres. During the extraction, the dispersion of the sorbents at milligram level was achieved by the in situ generation of carbon dioxide. The effervescence enhanced the interaction between the sorbent and the analytes. Langmuir and Freundlich models were used to evaluate adsorption processes. Under the optimum analytical conditions, the method showed good linearity (3-300μgL-1), acceptable precision (RSD <5%), low limits of quantification (1.02-2.98μgL-1) and satisfactory recoveries (90.05-99.85%). The proposed method was successfully applied to the analysis of alkaloids and flavonoids from Epimedii herba in cell culture fluids, rat urine and feces obtained at the different time intervals.

Flavonoids of Herba Epimedii Enhances Bone Repair in a Rabbit Model of Chronic Osteomyelitis During Post‐infection Treatment and Stimulates Osteoblast Proliferation in Vitro

Flavonoids are the active component of the Herba Epimedii (H. Epimedii), which is commonly used in Asia. This study is to investigate the effect of H. Epimedii on bone repair after anti‐infection treatment in vivo. The bioactive‐composition group of H. Epimedii (BCGE) contained four flavonoids with the total content of 43.34%. Rabbits with chronic osteomyelitis in response to injection with Staphylococcus aureus were treated with BCGE of 242.70 mg/kg/day intragastrically after vancomycin‐calcium sulphate treatment. Micro‐computerd tomography (CT), morphology, blood biochemistry and osteocalcin levels were assessed for effect evaluation. In addition, the rat calvarial osteoblasts infected with S. aureus were treated with vancomycin and BCGE. Cell viability, alkaline phosphatase activity, bone morphogenetic protein 2, Runt‐related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor‐κB ligand mRNA levels and protein expression were assessed. Our results indicated that BCGE promoted bone repair via increasing the bone mass, the volume of bone, promoting osteocalcin secretion after vancomycin‐calcium sulfate treatment. BCGE enhanced the cell proliferation, by regulating bone morphogenetic protein 2, runt‐related transcription factor 2, and osteoprotegerin/receptor activator of nuclear factor κ‐B ligand mRNA and protein expression to maintain the balance between bone formation and bone resorption. Therefore, BCGE is a potential adjuvant herbal remedy for the post‐infection treatment of chronic osteomyelitis. Copyright

A Network Pharmacology Approach to Determine the Active Components and Potential Targets of Curculigo Orchioides in the Treatment of Osteoporosis

Background Osteoporosis is a complex bone disorder with a genetic predisposition, and is a cause of health problems worldwide. In China, Curculigo orchioides (CO) has been widely used as a herbal medicine in the prevention and treatment of osteoporosis. However, research on the mechanism of action of CO is still lacking. The aim of this study was to identify the absorbable components, potential targets, and associated treatment pathways of CO using a network pharmacology approach. Material/Methods We explored the chemical components of CO and used the five main principles of drug absorption to identify absorbable components. Targets for the therapeutic actions of CO were obtained from the PharmMapper server database. Pathway enrichment analysis was performed using the Comparative Toxicogenomics Database (CTD). Cytoscape was used to visualize the multiple components-multiple target-multiple pathways-multiple disease network for CO. Results We identified 77 chemical components of CO, of which 32 components could be absorbed in the blood. These potential active components of CO regulated 83 targets and affected 58 pathways. Data analysis showed that the genes for estrogen receptor alpha (ESR1) and beta (ESR2), and the gene for 11 beta-hydroxysteroid dehydrogenase type 1, or cortisone reductase (HSD11B1) were the main targets of CO. Endocrine regulatory factors and factors regulating calcium reabsorption, steroid hormone biosynthesis, and metabolic pathways were related to these main targets and to ten corresponding compounds. Conclusions The network pharmacology approach used in our study has attempted to explain the mechanisms for the effects of CO in the prevention and treatment of osteoporosis, and provides an alternative approach to the investigation of the effects of this complex compound.

Efficient induction of antimicrobial activity with vancomycin nanoparticle-loaded poly(trimethylene carbonate) localized drug delivery system

Surgery and the local placement of an antibiotic are the predominant therapies to treat chronic osteomyelitis. Vancomycin-loaded N-trimethyl chitosan nanoparticles (VCM/TMC NPs) as a potential drug delivery system have high intracellular penetration and effective intracellular antibacterial activity. This study investigated the effects of a biocompatible material, poly(trimethylene carbonate) (PTMC), to increase the sustained effectiveness of an intracellular antibiotic and its potential application in antibiotic delivery. VCM/TMC NP-PTMC was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy to determine the morphology, stability and chemical interaction of the drug with the polymer. Further, the biodegradation, antibacterial activity, protein adsorption, cell proliferation and drug release characteristics were evaluated. In addition, a Staphylococcus aureus-induced osteomyelitis rabbit model was used to investigate the antibiotic activity and bone repair capability of VCM/TMC NP-PTMC. The results showed that the composite beads of VCM/TMC NPs followed a sustained and slow release pattern and had excellent antibacterial activity and a higher protein adsorption and cell proliferation rate than the VCM-PTMC in vitro. Furthermore, VCM/TMC NP-PTMC inhibits bacteria and promotes bone repair in vivo. Thus, VCM/TMC NP-PTMC might be beneficial in periodontal management to reduce the bacterial load at the infection site and promote bone repair.

Monosaccharide composition analysis of immunomodulatory polysaccharides by on‐line hollow fiber microextraction with high‐performance liquid chromatography

The monosaccharide compositions of functional polysaccharides are essential for structure elucidation and biological activity determination. A sensitive method based on on-line hollow-fiber liquid-phase microextraction with high-performance liquid chromatography has been established for the analysis of ten monosaccharide compositions (two uronic acids, two amino sugars and six neutral sugars) of the immunomodulatory polysaccharides. After derivatization, the sample was injected into the lumen of a hollow fiber immersed in butyl ether and separated by liquid chromatography. Under optimized conditions, the calibration curves were linear (r ≥ 0.9996) in the range of 10-2000 μmol L(-1) . The limits of detection were in the range of 0.04-1.58 μmol L(-1) , and the recoveries were in the range of 92.1-99.6%, which shows that the method is applicable to the analysis of the monosaccharide composition of various polysaccharides.

Front Cover: Osteoblast cell membrane chromatography coupled with liquid chromatography and time‐of‐flight mass spectrometry for screening specific active components from traditional Chinese medicines

A method using osteoblast membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry was developed to recognize and identify the specific active components from traditional Chinese medicines. Primary rat osteoblasts were used for the preparation of the stationary phase in the cell chromatography method. Retention components from the cell chromatography were collected and analyzed by liquid chromatography with time-of-flight mass spectrometry. This method was applied in screening active components from extracts of four traditional Chinese medicines. In total, 24 potentially active components with different structures were retained by osteoblast cell chromatography. There were five phenolic glucosides and one triterpenoid saponin from Curculigo orchioides Gaertn, two organic acids and ten flavonoids from Epimedium sagittatum Maxim, one phthalide compound and one organic acid from Angelica sinensis Diels, and two flavonoids and two saponins from Anemarrhena asphodeloides Bunge. Among those, four components (icariin, curculigoside, ferulaic acid, and timosaponin BII) were used for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation, alkaline phosphatase activity, levels of bone gla protein and collagen type 1, and promoted mineralized nodule formation. The developed method was an effective screening method for finding active components from complex medicines that act on bone diseases.

Icariin Enhances Bone Repair in Rabbits with Bone Infection during Post-infection Treatment and Prevents Inhibition of Osteoblasts by Vancomycin

Vancomycin is an effective antibiotic for treatment of bone infection caused by Staphylococcus aureus, however, a high local concentration of vancomycin might induce a delay in bone union. Icariin has been reported to suppress osteoclastogenes and promote osteogenesis. Our study aimed to investigate the effect of icariin on bone repair after anti-infection treatment in vivo and to explore the resisting effect of icariin on rat calvarial osteoblasts (ROBs) inhibited with high doses of vancomycin. Rabbits with bone infection of S. aureus were treated with implanted vancomycin-calcium sulfate (VCS) and icariin at 10.86 mg/kg/day for consecutive 8 weeks. Micro-CT, morphology, blood biochemistry were evaluated. In addition, ROBs were treated with vancomycin and icariin at different doses. Cell proliferation and differentiation capabilities, BMP2, Runx2, OPG, RANKL mRNA levels and protein expression were assessed. The results indicated that high dose of vancomycin significantly decreased bone mass and inhibited osteocalcin secretion; icariin increased these indicators compared with the single vancomycin treatment. Over 0.1 mg/mL of vancomycin inhibited the proliferation and differentiation of ROBs, while icariin resisted the inhibition of vancomycin by regulating cell cycle and promoting the Alkaline phosphatase (ALP) activity. Moreover, icariin promote bone formation by up-regulating BMP2/Runx2 and OPG/RANKL pathways. Icariin exhibited osteoplastic properties on osteoblasts that had been inhibited with high doses of vancomycin. Therefore, icariin is helpful for post-infection treatment of bone infection.

Effects of alkaloids from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts

Chronic osteomyelitis is primarily caused by infection with Staphylococcus aureus (S. aureus). Antibiotics are commonly administered; however, it is a challenge to promote bone healing. The aim of this study was to investigate the in vitro effects of alkaloids from the herbal remedy Sophora flavescens (ASF) on rat calvarial osteoblasts (ROBs) infected with S. aureus and healthy osteoclasts. Cell proliferation and alkaline phosphatase, interleukin‐6, and tumour necrosis factor‐α activity was measured in infected ROBs; tartrate‐resistant acid phosphatase was evaluated in osteoclasts via enzyme‐linked immunosorbent assay. The mRNA and protein expression levels of bone morphogenetic protein 2, runt‐related transcription factor 2, osteoprotegerin, and receptor activator of nuclear factor kappa‐B ligand were assessed in infected ROBs through reverse transcription‐polymerase chain reaction and western blotting analysis, respectively. Results indicated that ASF increased the viability of uninfected ROBs and infected ROBs treated with vancomycin via regulation of bone morphogenetic protein 2, runt‐related transcription factor, osteoprotegerin, and receptor activator of nuclear factor kappa‐B ligand mRNA and protein expression levels. In addition, the secretion of the inflammatory factor tumour necrosis factor‐α was decreased and alkaline phosphatase activity was increased, inhibiting the viability of osteoclasts and tartrate‐resistant acid phosphatase activity. Therefore, the herbal remedy ASF has potential as a new treatment for chronic osteomyelitis.