Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease that has become the third leading cause of death worldwide. Cycloastragenol (CAG), which is the genuine sapogenin of the main active triterpene saponins in Astragali radix, is a bioavailable pre-clinical candidate for chronic obstructive pulmonary disease (COPD), and it was investigated in our previous study. In order to progress medical research, it was first efficiently produced on a 2.5-kg scale via Smith degradation from astragaloside IV (AS-IV). Simultaneously, since the impurity profiling of a drug is critical for performing CMC documentation in pre-clinical development, a study on impurities was carried out. As these structures do not contain chromophores and possess weak UV absorption characteristics, HPLC-CAD and UPLC-LTQ-Orbitrap-MS were employed to carry out the quality control of the impurities. Then, column chromatography (CC), preparative thin-layer chromatography (PTLC), and crystallization led to the identification of 15 impurities from CAG API. Among these impurities, compounds 1, 4, 9, 10, 14, and 15 were elucidated via spectroscopic analysis, and 2-3, 5-8, and 11-13 were putatively identified. Interestingly, the new compounds 9 and 14 were rare 10, 19-secocycloartane triterpenoids that displayed certain anti-inflammatory activities against LPS-induced lymphocyte cells and CSE-induced MLE-12 cells. Additionally, a plausible structural transformation pathway of the degradation compounds from CAG or AS IV was proposed. The information obtained will provide a material basis to carry out the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided regarding the compounds with weak UV absorption characteristics.

Approaches to Configuration Determinations of Flexible Marine Natural Products: Advances and Prospects.

Flexible marine natural products (MNPs), such as eribulin and bryostatin, play an important role in the development of modern marine drugs. However, due to the multiple chiral centers and geometrical uncertainty of flexible systems, configuration determinations of flexible MNPs face great challenges, which, in turn, have led to obstacles in druggability research. To resolve this issue, the comprehensive use of multiple methods is necessary. Additionally, configuration assignment methods, such as X-ray single-crystal diffraction (crystalline derivatives, crystallization chaperones, and crystalline sponges), NMR-based methods (JBCA and Mosher's method), circular dichroism-based methods (ECCD and ICD), quantum computational chemistry-based methods (NMR calculations, ECD calculations, and VCD calculations), and chemical transformation-based methods should be summarized. This paper reviews the basic principles, characteristics, and applicability of the methods mentioned above as well as application examples to broaden the research and applications of these methods and to provide a reference for the configuration determinations of flexible MNPs.

MS/MS-Based Molecular Networking: An Efficient Approach for Natural Products Dereplication.

Natural products (NPs) have historically played a primary role in the discovery of small-molecule drugs. However, due to the advent of other methodologies and the drawbacks of NPs, the pharmaceutical industry has largely declined in interest regarding the screening of new drugs from NPs since 2000. There are many technical bottlenecks to quickly obtaining new bioactive NPs on a large scale, which has made NP-based drug discovery very time-consuming, and the first thorny problem faced by researchers is how to dereplicate NPs from crude extracts. Remarkably, with the rapid development of omics, analytical instrumentation, and artificial intelligence technology, in 2012, an efficient approach, known as tandem mass spectrometry (MS/MS)-based molecular networking (MN) analysis, was developed to avoid the rediscovery of known compounds from the complex natural mixtures. Then, in the past decade, based on the classical MN (CLMN), feature-based MN (FBMN), ion identity MN (IIMN), building blocks-based molecular network (BBMN), substructure-based MN (MS2LDA), and bioactivity-based MN (BMN) methods have been presented. In this paper, we review the basic principles, general workflow, and application examples of the methods mentioned above, to further the research and applications of these methods.

Evaluation of pyrrolidine-based analog of jaspine B as potential SphK1 inhibitors against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitise, and its pathogenesis is complicated. Sphingosine-1-phosphate (S1P) is a lipid produced by sphingosine kinase 1 and 2 (SphK1/2), which participate in some of most-spread skeletal diseases such as rheumatoid arthritis or osteoarthritis. To explore the anti-inflammatory activity of 2-epi-jaspine B analogs as SphKs inhibitors, we used LPS-induced rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) as the research object to evaluate the anti-inflammatory activity of 16 2-epi-jaspine B analogs and the newly synthesized salt CHJ01. We found that 2-epi-jaspine B analog CHJ01 in hydrochloride salt form has excellent SphK1 inhibitory effect and better anti-RA effect. CHJ01 showed an anti-inflammatory effect similar to that of MTX in vitro, its IC50 value is 8.64 µM. Moreover, the anti-RA effect of CHJ01 was also studied by using a Complete Freund's Adjuvant (CFA)-induced arthritis (AIA) in a rat mode. Pharmacological experiments show that CHJ01 can help to significantly improve the symptoms of rheumatoid arthritis by reducing the swelling volume, arthritis score, spleen index and the level of IL-1ß, TNF-α, IL-6 of AIA rats. Therefore, CHJ01 holds high potential for the treatment of RA.

Endothelial ZEB1 promotes angiogenesis-dependent bone formation and reverses osteoporosis.

Recent interest in the control of bone metabolism has focused on a specialized subset of CD31hiendomucinhi vessels, which are reported to couple angiogenesis with osteogenesis. However, the underlying mechanisms that link these processes together remain largely undefined. Here we show that the zinc-finger transcription factor ZEB1 is predominantly expressed in CD31hiendomucinhi endothelium in human and mouse bone. Endothelial cell-specific deletion of ZEB1 in mice impairs CD31hiendomucinhi vessel formation in the bone, resulting in reduced osteogenesis. Mechanistically, ZEB1 deletion reduces histone acetylation on Dll4 and Notch1 promoters, thereby epigenetically suppressing Notch signaling, a critical pathway that controls bone angiogenesis and osteogenesis. ZEB1 expression in skeletal endothelium declines in osteoporotic mice and humans. Administration of Zeb1-packaged liposomes in osteoporotic mice restores impaired Notch activity in skeletal endothelium, thereby promoting angiogenesis-dependent osteogenesis and ameliorating bone loss. Pharmacological reversal of the low ZEB1/Notch signaling may exert therapeutic benefit in osteoporotic patients by promoting angiogenesis-dependent bone formation.

Eight new cucurbitane triterpenoids from "Xue Dan," the roots of Hemsleya pengxianensis.

Eight new natural products (four new cucurbitane aglycones, hemslepencins A-D (1-4), four new cucurbitane glucosides, hemslepensides F-I (5-8), along with seven known compounds (9-15), were isolated from the roots of Hemsleya pengxianensis. The structures of 1-8 were elucidated using IR, HRESIMS, and NMR. Compound 3 exhibited cytotoxic activity against the human cancer cell lines.

In vitro evaluation of marrow clot enrichment on microstructure decoration, cell delivery and proliferation of porous titanium scaffolds by selective laser melting three-dimensional printing.

Titanium alloy is a clinically approved material for bone substitution. Although three-dimensional printing (3DP) fabrication technique can build up porous Ti scaffolds with the designed shape and microstructure, the biomechanical performance of 3DP Ti scaffolds still need to be improved to increase the reliability of osseointegration capacity. To address this issue, rabbit bone marrow clot (MC) is used to modify 3DP Ti scaffolds by stem cell delivery and microenvironment decoration inside the pores of these scaffolds. Moreover, 3DP Ti scaffolds were built up using selective laser melting, and 3DP MC-Ti scaffolds were constructed through the enrichment of MC with Ti scaffolds in vitro. Results demonstrated that the obtained 3DP Ti scaffolds in current study has an average modulus of elasticity (ME) at 1294.48 MPa with average yield strength of 33.154 MPa. For MC-Ti scaffolds, MC enrichment obstructs the pores of 3DP scaffolds due to the large amount of fibrin and erythrocytes and leads to a decrease in ratio of live cells at 1-week culture. Cell proliferation and osteogenic differentiation performance of MC-Ti scaffolds were promoted with porous recanalization in the later 3 weeks. After 2 weeks in vitro culture, fivefold of cell number in MC-Ti scaffolds were observed than bone marrow-derived mesenchymal stem cell-seeded Ti scaffolds. Compared to Ti scaffolds, fourfold of deoxyribonucleic acid content, type I collagen-α1, osteocalcin, and alkaline phosphatase expression in MC-Ti scaffolds were observed after 4 weeks in vitro culture. Results suggested that the combination with MC is a highly efficient method that improves the biological performance of Ti scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2245-2253, 2018.

Application of 3D printing rapid prototyping-assisted percutaneous fixation in the treatment of intertrochanteric fracture.

The aim of the present study was to investigate the application of 3D printing (3DP) rapid prototyping (RP) technique-assisted percutaneous fixation in the treatment of femoral intertrochanteric fracture (ITF) using proximal femoral nail anti-rotation (PFNA). A total of 39 patients with unstable ITF were included in the current study. Patients were divided into two groups: 19 patients were examined using computed tomography scanning and underwent PFNA with SDP-RP whereas the other 20 patients underwent conventional PFNA treatment. Anatomical data were converted from the Digital Imaging and Communications in Medicine format to the stereolithography format using M3D software. The 3DP-RP model was established using the fused deposition modeling technique and the length and diameter of the main screw blade was measured during the simulation. The postoperative femoral neck-shaft angle (NSA), surgery duration, intraoperative and postoperative blood loss, and the duration of hospital stay were recorded and compared with the corresponding values in conventional surgery. No significant differences were observed in mean PFNA size between the implants used and the preoperative planning estimates. It was demonstrated that the 3DP-RP assisted procedure resulted in more effective reduction of the NSA. Furthermore, patients undergoing 3DP-RP experienced a significant reduction in duration of surgery (P<0.01), as well as reductions in intraoperative (P=0.02) and postoperative (P=0.03) blood loss, compared with conventional surgery. At 6 months post-surgery, no cases of hip varus/vague deformities or implant failure were observed in patients that underwent either the 3DP-RP-assisted or conventional procedure. The results of the present study suggest that the 3DP-RP technique is able to create an accurate model of the ITF, which facilitates surgical planning and fracture reduction, thus improving the efficiency of PFNA surgery for ITFs.

[Accurate osteotomy assisted by individualized navigation templates for the treatment of children cubitus varus].

OBJECTIVE: To investigate the feasibility and accuracy of a new navigation template for osteotomy in cubitus varus based on computer assistant design and 3D printing technology. METHODS: The preoperative CT images of 15 children with cubitus varus from June 2015 to June 2016 were collected. According to the above data, the individual osteotomy navigate template match the distal humerus was designed by the software and printed by the 3D printer. Accurate osteotomy was performed with the assistant of the navigate template in the operation. Internal fixation of the osteotomy site was performed with 2 Kirschner wires. After surgery, a long arm plaster was applied with 20° of elbow flexion. All the patients underwent radiographic and clinical evaluations before surgery and at the follow-up examination. RESULTS: During the operation, the navigate template with the individual design of 3D printing technology matched the bony markers of distal humerus. Accurate and simple osteotomy were performed along the resected surface of the navigation template. None of the cases required any kinds of revision surgery or had any complaint of cosmetic appearance. Average union time was 6.7 weeks(ranged, 6 to 8 weeks). Twelve patients got an excellent result and 2 got a good result according to the criteria described by Bellemore. There were no cases with complications of infection or ulnar nerve palsy or joint stiffness. CONCLUSIONS: With the help of 3D printing technology, the accurate osteotomy in cubitus varus assisted by individualized navigate template can be realized. This technology can restore normal anatomical structure of the elbow joint to the greatest extent. It is worthy of popularization and application.

[Comparative pharmacokinetics of syringin, eleutheroside E and isofraxidin in rat plasma after intravenous administration of each monomer and Ciwujia injection].

To compare the pharmacokinetics of syringin, eleutheroside E and isofraxidin after intravenous administration of each monomer and Ciwujia injection. Twenty-four Sprague-Dawley rats were randomly divided into four groups and intravenously administrated with syringin, eleutheroside E, isofraxidin, and Ciwujia injection, respectively. The concentrations of the three components in rat plasma were determined by LC-MS/MS. DAS 2.0 software was applied to calculate the pharmacokinetic parameters while the SPSS 17.0 software was used for statistical analysis. Significant difference (P < 0.05) was found between each monomer and the injection on the main pharmacokinetic parameters such as AUC, CL and t1,/2. Compared with the injection, the group treated with the syringin has obvious decrease in AUC, and increase in CL while the group treated with eleutheroside E has obvious increase in AUC, and decrease in CL The t1/2 of isofraxidin was prolonged in Ciwujia injection. Pharmacokinetic characters of the ingredients in the injection varied greatly from the monomer. Other constituents in the injection may have an impact on the pharmacokinetic profiles of these three components.

[Study on in vivo pharmacokinetics of cucurbitacin injection in rats].

To establish a method for the determination of cucurbitacin in plasma samples, in order to study the in vivo pharmacokinetic characteristics of cucurbitacin in rats. Rats were intravenously injected with cucurbitacin. With diphenhydramine as the internal standard (IS), the plasma concentrations of cucurbitacin in rat plasma at different time points were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). With electrospray ionization source, the positive ion detection in the multiple reaction monitoring mode was conducted to determine the ion-pairs for target compound and IS were m/z 503.2/113.1 and m/z 256.0/167.2, respectively. Agilent ZOBAX SB-C18 column (2.1 mm x 50 mm, 1.8 microm) was adopted and eluted with methanol and 0.1% formic acid (55:45), and the flow rate was 0.2 mL x min(-1). DAS 2.0 software was applied to fit the blood concentration and calculate corresponding pharmacokinetic parameters. The rats were intravenously injected with cucurbitacin at the concentration of 3.0 mg x kg(-1). The target blood quality concentration show good linear relations within the range of 10.5-3 150 microg x L(-1) (R2 = 0.996), the lower limit of the standard curve was 10.5 microg x L(-1), and the signal to noise ratio S/N = 12. Intra- and inter-day precisions RSD was less than 6.9% and 14%, respectively; The accuracy RE ranged between 0.20% and 3.7%; The extraction recoveries ranged between 92.7% and 97.1%. Regarding the pharmacokinetic parameters of tail intravenous injection of cucurbitacin, AUC (0-t) was (811.615 +/- 111.578) microg x h x L(-1), (t1/2) was (1.285 +/- 1.390) h, CL was (3.627 +/- 0.487) L x h x kg(-1), and V(d) was (6.721 +/- 7.429) L x kg(-1). In this study, researchers established a simple, accurate, sensitive and highly specific method for determining the blood concentration of cucurbitacin, and reported the in vivo pharmacokinetic characteristics of cucurbitacin in rats for the first time.

Biomechanical effects of semi-constrained integrated artificial discs on zygapophysial joints of implanted lumbar segments.

This study aimed to optimize the design and application of semi-constrained integrated artificial discs (SIADs) using a finite element (FE) analysis following implantation, wherein the zygapophysial joints of the segment were biomechanically reconstructed. An FE model of the L4-L5 segment was constructed. Variations in the stresses on the discs and zygapophysial joints were observed during 5° anteflexion, 5° extension and 5° rotation under the 400-N applied axial load. Stresses and load translation analyses of the discs and zygapophysial joints were conducted during anteflexion, extension and rotation under the 400-N applied axial load. Following implantation of the lumbar segments, the stresses on the SIAD zygapophysial joints were not significantly different from those of physiological discs during anteflexion, and these were both marginally greater compared with those of non-constrained artificial discs (NADs). During extension, the increase in the stress on the SIAD zygapophysial joints was less than that on NAD zygapophysial joints. Stresses on the NAD zygapophysial joints were higher than those on SIAD and physiological discs during rotation. The stress on the SIAD zygapophysial joints was not significantly different from that on physiological discs during rotation. For SIADs and NADs, the stresses on the zygapophysial joints and the displacements of the discs were greater compared with those of the physiological discs during extension. The SIADs affected the variations in the stresses on the implanted segment more than the NADs, and the SIADs protected the zygapophysial joints of the implanted segment to a higher degree than the NADs.

Oldhamianoside II, a new triterpenoid saponin, prevents tumor growth via inducing cell apoptosis and inhibiting angiogenesis.

Oldhamianoside II is a new triterpenoid saponin that was isolated from the roots of Gypsophila oldhamiana. The present study aims to investigate the potential inhibitory activity of oldhamianoside II on tumor growth using an S180 tumor implantation mouse model. Oldhamianoside II at doses of 5.0 and 10.0 mg/kg was given with intraperitoneal injection for 10 days following subcutaneous inoculation of S180 tumor cells in anterior flank of mice. The tumor growth, the cell apoptosis, the microvessel density (MVD) in S180 tumors, the tumor cell viability, the tubular formation in vitro, and migration of tumor cells were examined. The expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and cyclooxygenase-2 (COX-2) was determined to analyze the associated mechanisms. The results showed that oldhamianoside II potently inhibited tumor cell viability in vitro. In addition, oldhamianoside II delayed tumor growth in anterior flank, induced S180 cell apoptosis, and reduced the MVD. Oldhamianoside II was also demonstrated to decrease the number of tubular structure and vessel formation in HUVEC cultures and chick embryo chorioallantoic membrane (CAM) model, respectively. Further study indicated that oldhamianoside II reduced the expression of VEGF, bFGF, and COX-2 in tumor sections. Moreover, oldhamianoside II inhibited the activity of migration and penetration to Matrigel of SGC7901 tumor cells in scratch wound and transwell chamber. In conclusion, our work defines oldhamianoside II, a new triterpenoid saponin, as a novel compound that can effectively inhibit S180 tumor growth, induce tumor cell apoptosis, prevent tumor angiogenesis, and inhibit cancer cell migration, suggesting that oldhamianoside II is a potential drug candidate for the treatment of cancer and for the prevention of metastasis.

A new chromone from Angelica polymorpha with cytotoxic activity.

A new chromone polymorchromone B was isolated and characterized from the roots of Angelica polymorpha Maxim, a commonly used traditional Chinese medicine. On the basis of spectroscopic analysis, including IR, HR-ESI-MS, 1D and 2D NMR spectral data, and hydrolysis followed by chromatographic analysis, the structure of the new chromone was elucidated as 5-hydroxy-2-[(angeloyloxy)methyl]-2'-(dimethyl)-3'-(2S,3S-epoxy-2-methylbutanoate)-dihydropyran [3',2':6,7]chromone. Moreover, it was found that polymorchromone B showed remarkable cytotoxic activity against A-549 cell lines.

Two new 4-arylcoumarins from the seeds of Calophyllum polyanthum.

Two new 4-arylcoumarins, 7,4'-dihydroxy-6,8-dimethoxy-4-phenylcoumarin (1) and 7-hydroxy-6,8,4'-trimethoxy-4-phenylcoumarin (2), together with four known compounds were isolated from the seeds of Calophyllum polyanthum. The structures of the new compounds were determined by extensive spectroscopic analyses, and the structure of compound 2 was confirmed by X-ray crystallography analysis. Both new compounds exhibited significant cell protective activities against H(2)O(2)-induced human umbilical vein endothelial cell damage.

New flavonoids with 2BS cell proliferation promoting effect from the seeds of Trigonella foenum-graecum L.

Ten flavonoids were isolated from the ethyl acetate-soluble fraction of the ethanolic extract of the seeds of Trigonella foenum-graecum and their structures were elucidated on the basis of spectroscopic methods to be 5,7,3'-trihydroxy-5'-methoxylisoflavone (1), biochanin A (2), formononetin (3), irilone (4), tricin (5), daidzein (6), calycosin (7), orientin-2''-O-p-trans-coumarate (8), vitexin-2''-O-p-trans-coumarate (9), and tricin-7-O-beta-D: -glucopyranoside (10). Compounds 1 and 8 are new flavonoids, and 8 and 9 strongly promoted 2BS cell proliferation induced by H(2)O(2).

Effects of a new shape-memory alloy interspinous process device on pressure distribution of the intervertebral disc and zygapophyseal joints in vitro.

OBJECTIVE: To quantify the pressure distribution of lumbar intervertebral discs and zygapophyseal joints with different degrees of distraction of the interspinous processes by using a new shape-memory interspinous process stabilization device, and to research the relationship between changing disc and zygapophyseal joint loads and the degree of distraction of interspinous processes, and thus optimize usage of the implant. METHODS: Six cadaver lumbar specimens (L(2)-L(5)) were loaded. The loads in disc and zygapophyseal joints were recorded at each L(3-4) disc level. Implants with different spacer heights were then placed by turn and the pressure measurements repeated. RESULTS: An implant with 10 mm spacer height does not significantly share the load. A 12 mm implant reduces the posterior annulus load, and meanwhile decreases the zygapophyseal joints pressure, but only in extension. A 14 mm implant shares the loads of posterior annulus, nucleus, and zygapophyseal joints in extension and the neutral position, but slightly increases the anterior annulus' load. Though 16-20 mm implants do decrease the loads in the posterior annulus and zygapophyseal joints, the anterior annulus' load was apparently increased. CONCLUSION: Different degrees of distraction of the interspinous processes lead to different load distribution on the intervertebral disc. The implant tested is not appropriate in cases of serious spinal stenosis because of the contradiction that, while over-distraction of the interspinous processes decreases the posterior annulus and the zygapophyseal joints load and distracts the intervertebral foramina, it leads to a marked increase in the load of the anterior annulus, which is recognized to accelerate disc degeneration.

[Chemical constituents of Sparganium stoleniferum].

To study the chemical constituents of Sparganium stoleniferum Buch. -Ham, various chromatographic techniques were used to separate and purify the chemical constituents. Their physicochemical properties and spectral data were used to elucidate the structures. Five compounds have been isolated by using silica gel column chromatography. They are beta-sitosterol plamitate (I), SanLeng diphenyllactone (II), SanLeng diphenylacetypene (III), 6,7,10-trihydroxy-8-octadecenoic acid (IV) and daucosterol plamitate (V). Compound II, III are two new compounds.

[Effect of preparation on the major chemical constituents of Polygonum multiflorum].

OBJECTIVE: To study the dynamic changes of Emodin, Physcion, Emodin-8-O-beta-D-glucopyranoside, Physcion-8-O-beta-D-glucopyranoside and 2,3,5,4'-tetrahydtoxysilbene-2-O-beta-D-glucopyranoside in Polygonum multiflorum during preparation and analyze their changing laws. METHODS: Five major constituents before and after preparation were determined by RP-HPLC methods. RESULTS: The five ingredients decreased significantly after preparation. CONCLUSION: The major chemical constituents of Polygonum multiflorum decrease significantly, suggesting the necessarity of prepared Polygonum multiflorum in clinical application worth studying.