Two new chemical constituents from the rhizomes of Actaea dahurica.

A new phenylpropanoid allopyranoside (1) and a new indolinone alkaloid (2) were isolated from the rhizomes of Actaea dahurica (syn. Cimicifuga dahurica). The structures of those two compounds were deduced as cimicifugaside F (1) and 3E,11E-(3-methyl-2-butenylidene acid)-2-indolinone-1-O-ß-d-glucopyranoside (2) by detailed analysis of their MS, 1D and 2D NMR data and comparison with literatures. Additionally, the isolates were evaluated for their inhibitory effects on the production of NO by LPS-stimulated RAW 264.7 macrophages.

Systematically identifying the anti-inflammatory constituents of Cimicifuga dahurica by UPLC-Q/TOF-MS combined with network pharmacology analysis.

Cimicifuga dahurica (Turcz.) Maxim, which is also regarded as the main origin of "Shengma" in the Chinese Pharmacopoeia, has been used as a cooling and detoxification agent for thousands of years. Our previous phytochemical investigations of C. dahurica extracts (CDEs) led to the isolation of a series of 9,19-cycloalkane triterpenoids and phenolic acids showing a potential anti-inflammatory activity. However, the chemical profiling of CDEs and the material basis of its anti-inflammatory effect in vivo has not been clarified. In the present study, the CDE chemical profile and prototype components in rat plasma were identified via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. As a result, a total of 106 components were identified or tentatively characterized in CDEs, including 54 triterpenoids, 35 phenolic acids, eight amides and nine other type constituents (39 compounds were confirmed with the reference standards). In addition, 20 prototype components (15 triterpenoids and five phenolic acids) were identified in rat plasma, which potentially related to the anti-inflammatory effects of CDEs. Moreover, the anti-inflammatory activities of the main prototype components were further evaluated by their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in lipopolysaccharide-stimulated RAW264.7 cells, which indicated that 9,19-cycloalkane triterpenoids may play an anti-inflammatory role by down-regulating the expression of iNOS.

Three new cycloart-7-ene triterpenoid glycosides from Cimicifuga dahurica and their anti-inflammatory effects.

Ten cycloart-7-ene triterpenoid glycosides, including three new compounds (1-3), were isolated from the roots of Cimicifuga dahurica. Their structures were elucidated on the basis of extensive spectroscopic analyses, chemical methods and comparison with literatures. In addition, the isolates were evaluated for their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in LPS-stimulated RAW 264.7 macrophages. The results showed that compounds 3, 5, 6, 7 and 8 can reduce the release of NO in a dose-dependent manner. Mechanistically, Western blot analysis indicated that the NO inhibitory effects relied on down-regulating the expression of iNOS, and partially associated with lowering the expression of COX-2.

Diarylheptanoid analogues from the rhizomes of Zingiber officinale and their anti-tumour activity.

Eight previously undescribed diarylheptanoids (1-8), together with fifteen known analogues (9-23), were isolated from the rhizomes of Zingiber officinale. Their structures were unambiguously determined by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. It is worth mentioning that 1-3 are the first reported structures of diaryl ether heptanoids in Z. officinale, whereas 15-17 were isolated from Zingiber for the first time. Furthermore, a gene enrichment analysis of the interacting targets indicated that diarylheptanoids were mainly associated with the anti-tumor activity by affecting DNA damage signaling pathway. The results showed that 6, 16-19 had remarkable inhibitory effects against five tumor cell lines (A549, HepG2, HeLa, MDA-MB-231, and HCT116) with IC50 values ranging from 6.69-33.46 µM, and showed down-regulating the expression of ATR (ataxia telangiectasia mutated and RAD3-related) and CHK1 (checkpoint kinase 1) levels in HCT116 and A549 cell lines. Our studies not only enrich the structural diversity of diarylheptanoids in nature, but also discover several natural compounds for anti-tumor agents.

EGCG targeting Notch to attenuate renal fibrosis via inhibition of TGFß/Smad3 signaling pathway activation in streptozotocin-induced diabetic mice.

Renal fibrosis is a characteristic of diabetic nephropathy, which is a serious complication of diabetes. It has been reported that (-)-epigallocatechin gallate (EGCG) attenuates renal fibrosis. However, the molecular mechanism of regulation by EGCG in this process remains unclear. Previous studies showed that abnormal activation of Notch signaling contributes to the development of renal fibrosis. Previous studies have demonstrated that EGCG attenuates Notch1 expression. In this study, we found that the levels of fibronectin and Notch1 expression were decreased in human embryonic kidney cells after treatment with EGCG. We also observed that the type II transforming growth factor beta receptor (TGFßRII) and Smad3 pathway were inhibited in kidney cells by treatment with EGCG. In the diabetic kidney, we found that the activation of Notch signaling was attenuated by administration of EGCG. Moreover, TGFßRII and Smad3 phosphorylation could be inhibited by treatment with EGCG in the kidney. These results indicated that EGCG may improve renal fibrosis by targeting Notch via inhibition of the TGFß/Smad3 pathway in diabetic mice. Our findings provide insight into the therapeutic strategy for diabetes-induced renal fibrosis, and suggest EGCG to be a novel potential medicine for the treatment of chronic kidney disease in patients with diabetes.

Structurally diverse sesquiterpenoids from the aerial parts of Artemisia annua (Qinghao) and their striking systemically anti-inflammatory activities.

Thirteen new sesquiterpenoids, arteannoides F-R (1-13), along with 13 known analogues (14-26), were isolated from the dried aerial parts of Artemisia annua L. Their structures, including absolute configurations, were unambiguously determined by a combination of physical data analyses (HRESIMS, 1D and 2D NMR, and ECD) as well as the crystal structures of 1, 5, 6, 15, 19, and 23. Among the isolated compounds, 1 features an unusual 11-oxatricyclo[6.2.1.04,9]undecan-2-ene ring system, 5 possesses an uncommon 4,11-ether bridged tricyclic framework, whereas 6 is a new eudesmane-type sesquiterpenoid formed via rearrangement of its carbon backbone. The systemically anti-inflammatory activities of all isolates were evaluated by measuring their inhibitory effects on PGE2, NO, TNF-α, and IL-6 production in LPS-stimulated RAW 264.7 macrophages. Moreover, the structure activity relationships of some compounds are summarized, this study will provide new structural templates for discovering potential anti-inflammatory agents.

Magnesium and vitamin C supplementation attenuates steroid-associated osteonecrosis in a rat model.

Magnesium (Mg)-based biometal attracts clinical applications due to its biodegradability and beneficial biological effects on tissue regeneration, especially in orthopaedics, yet the underlying anabolic mechanisms in relevant clinical disorders are lacking. The present study investigated the effect of magnesium (Mg) and vitamin C (VC) supplementation for preventing steroid-associated osteonecrosis (SAON) in a rat experimental model. In SAON rats, 50 mg/kg Mg, or 100 mg/kg VC, or combination, or water control was orally supplemented daily for 2 or 6 weeks respectively. Osteonecrosis was evaluated by histology. Serum Mg, VC, and bone turnover markers were measured. Microfil-perfused samples prepared for angiography and trabecular architecture were evaluated by micro-CT. Primary bone marrow cells were isolated from each group to evaluate their potentials in osteoblastogenesis and osteoclastogenesis. The mechanisms were tested in vitro. Histological evaluation showed SAON lesions in steroid treated groups. Mg and VC supplementation synergistically reduced the apoptosis of osteocytes and osteoclast number, and increased osteoblast surface. VC supplementation significantly increased the bone formation marker PINP, and the combination significantly decreased the bone resorption marker CTX. TNFα expression and oxidative injury were decreased in bone marrow in Mg/VC/combination group. Mg significantly increased the blood perfusion in proximal tibia and decreased the leakage particles in distal tibia 2 weeks after SAON induction. VC significantly elevated the osteoblast differentiation potential of marrow cells and improved the trabecular architecture. The combination supplementation significantly inhibited osteoclast differentiation potential of marrow cells. In vitro study showed promoting osteoblast differentiation effect of VC, and anti-inflammation and promoting angiogenesis effect of Mg with underlying mechanisms. Mg and VC supplementation could synergistically alleviate SAON in rats, indicating great translational potentials of metallic minerals for preventing SAON.

Safety and efficacy of cyclooxygenase-2 inhibition for treatment of primary hypertrophic osteoarthropathy: A single-arm intervention trial.

BACKGROUND: Primary hypertrophic osteoarthropathy (PHO) is a rare disease involving joint, bone and skin. Two underlying genes responsible for this disease-hydroxyprostaglandin dehydrogenase (HPGD) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1)-are both associated with aberrant accumulation of prostaglandin E2 (PGE2). Cyclooxygenase-2 (COX-2) is a key enzyme in PGE2 synthesis. This study was intended to evaluate the safety and efficacy of COX-2 inhibitor in the treatment of PHO. METHODS: We recruited patients presenting to Peking Union Medical Hospital between January 2009 and December 2016 who were diagnosed with PHO. Participants were given the COX-2 inhibitor etoricoxib (60 mg once daily) and followed up for 9 months. Gene analysis was performed at baseline. The following data were collected at baseline and during treatment: visual analogue score (VAS), volume of the distal middle finger (VDMF), knee joint circumference (KJC), serum and urinary levels of prostaglandin E2 (PGE2) and PGE metabolite (PGE-M) and serum levels of inflammatory markers. RESULTS: A total of 27 patients were recruited, including seven patients with PHO type I (PHOAR1) carrying HPGD gene mutations and 20 patients with PHO type II (PHOAR2) carrying SLCO2A1 gene mutations. After treatment with etoricoxib, the majority of patients experienced resolution of symptoms including pachydermia (60.9%), joint swelling (100%), digital clubbing (74.1%) and hyperhidrosis (55.0%). In both the PHO subtypes, serum and urinary levels of PGE2 were elevated at baseline and declined sharply upon treatment. For PHOAR1 patients, serum and urinary PGE-M levels were relatively low and demonstrated minimal response to COX-2 inhibition. Among PHOAR2 patients, mean serum and urinary levels of PGE-M presented at a high level at baseline and were normalized after 3 months of treatment. No severe adverse effects were reported during the study period. CONCLUSIONS: We found COX-2 inhibitor to be safe and effective for the treatment of PHO in our cohort. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The underlying genes responsible for PHO suggest COX inhibitor as potential therapy, and our study demonstrates the efficacy and safety of this treatment.

New phenylpropanoid allopyranosides from the rhizomes of Cimicifuga dahurica.

Five new phenylpropanoid allopyranosides (1-5), along with five known compounds (6-10) were isolated from the rhizomes of Cimicifuga dahurica. Their structures were established by means of spectroscopic analyses and chemical methods, as well as comparison with literatures. The anti-inflammatory activities of all isolates were evaluated. Compounds 6, 9 and 10 exhibited inhibitory effects on PGE2 production in LPS stimulated RAW 264.7 macrophages with IC50 values of 19.72, 6.33 and 39.90 µM, respectively.

Phenolic acids and their glycosides from the rhizomes of Cimicifuga dahurica.

Phytochemical study on rhizomes of Cimicifuga dahurica resulted in the isolation of nine new neolignan and phenylpropanoid glycosides, cimicifugasides A-E (1, 2, 7-9), cimicifugamides B-D (3-5), shomaside G (6) along with four known compounds (10-13). Their structures were identified by extensive spectroscopic analyses (1D-, 2D-NMR, MS, CD, IR, UV) and chemical methods. Their anti-inflammatory potentials were evaluated by measuring their effects on PGE2 production of LPS-stimulated RAW264.7 cells, and compounds 12 and 13 showed moderate anti-inflammatory activities.

Lonimacranaldes A-C, three iridoids with novel skeletons from Lonicera macranthoides.

Lonimacranaldes A and B (1 and 2), along with one biogenetically related intermediate, lonimacranalde C (3), were isolated from the flower buds of Lonicera macranthoides. Characterized by an iridoid structure and an additional C-6 unit with an aldehyde group, compounds 1 and 2 are the first examples of hybrid iridoids possessing an unexpected 6/5/6 fused tricyclic ring system, while compound 3 serves as an important precursor for their generation. The structures of lonimacranaldes A-C (1-3) were revealed by extensive spectroscopic and X-ray diffraction analyses. A plausible biogenetic pathway for them was proposed. Compound 3 showed anti-inflammatory activities by inhibiting the production of IL-6 on LPS-induced RAW 264.7 cells with an IC50 value of 6.33 µM.

Steroid-associated osteonecrosis animal model in rats.

OBJECTIVE: Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS). METHODS: Sixteen 24-week-old male Sprague-Dawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy X-ray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination. RESULTS: Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction. CONCLUSION: The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON.

Solvent-responsive floating liquid crystalline-molecularly imprinted polymers for gastroretentive controlled drug release system.

Liquid crystalline-molecularly imprinted polymer (LC-MIP) particles were first found to possess the floating behavior on the aqueous medium. Combined with molecular recognition, the LC-MIP was fabricated as a novel class of the controlled-release gastric retentive DDS. The LC-MIP was made using co-polymerization of methacrylic acid, 4-methyl phenyl dicyclohexyl ethylene (LC monomer with vinyl group), and ethylene glycol dimethacrylate with S-amlodipine (S-AML) as model template drug. The optimum condition of the preparation of LC-MIP has been obtained relying on release behaviors of S-AML from the LC-MIP. The surface morphology of LC-MIP and three corresponding control samples, i.e., template-free LC-NIP, LC-free MIP, and LC-free NIP, were studied. Applying the LF model for the binding isotherm, imprinting factors was 2.80 for the LC-MIP with the crosslinking degree of 20.0%, whereas 6.70 for the LC-free MIP with high levels of crosslinker (80.0%). Furthermore, the phase transition behaviors of LC-based particles as well as drug-loaded LC elastomers were measured by a differential scanning calorimeter and the formed hydrogen bonding between S-AML and LC-MIP was demonstrated by FT-IR spectra. In vivo imaging experiment proved that the floating LC-MIP had significantly longer gastric residence time (>60min) than the non-floating MIP reference (<30min). In vivo pharmacokinetic study showed a plateau region between 1.5 and 22h on the plasma concentration from the LC-MIP. In spite of lower imprinting factor, the relative bioavailability of the gastro-floating LC-MIP was 180.5%, whereas only 111.7% of the LC-free MIP. As a conclusion, the LC-MIPs indicated potentials for oral administration due to the innovative combination of floating and controlled release properties.

[Clinical features of hereditary distal renal tubular acidosis and SLC4A1 gene mutation].

OBJECTIVE: To study the clinical features of two families with distal renal tubular acidosis (dRTA) and mutations in the pathogenic gene SLC4A1. METHODS: Family investigation, medical history collection, and measurement of biochemical parameters were performed to analyze the clinical phenotype and genetic characteristics of dRTA. Direct sequencing was used to detect SLC4A1 gene mutations. RESULTS: Three patients in these two families (two of them were mother and son) were diagnosed with dRTA with typical clinical features, including short stature, metabolic acidosis, alkaline urine, hypokalemia, and nephrocalcinosis. SLC4A1 gene analysis showed that all the three patients had a pathogenic missense mutation R589H (c.1766G>A). The child in family 1 had a de novo mutation of SLC4A1, and the child in family 2 had an SLC4A1 gene mutation inherited from the mother, which met the characteristic of autosomal dominant inheritance. CONCLUSIONS: This study reports the R589H mutation in SLC4A1 gene in families with hereditary dRTA for the first time in China. Clinical physicians should perform gene detection for patients suspected of hereditary dRTA to improve the diagnosis and treatment of this disease.

A Common Mutation and a Novel Mutation in the HPGD Gene in Nine Patients with Primary Hypertrophic Osteoarthropathy.

Primary hypertrophic osteoarthropathy (PHO) is a hereditary bone disease characterized by digital clubbing, periostosis, and pachydermia. The HPGD gene encoding 15-prostaglandin dehydrogenase and SLCO2A1 encoding one type of prostaglandin transporter were found to be responsible for PHO. Mutations of either gene would lead to increased level of prostaglandin E2 (PGE2), which might contribute to the constellation of the symptoms. The aim of the study was to analyze the HPGD gene and the clinical characteristics in nine patients with the diagnosis of PHO. Nine patients, (eight males and one female) including two siblings and seven sporadic cases, were enrolled in the study. Clinical features were summarized, and blood and urine samples were collected. Sanger method was used to sequence the HPGD gene to detect mutations. Urinary PGE2 and prostaglandin metabolite (PGE-M) levels for each patient were measured and compared to the healthy controls. A recurrent c.310_311delCT mutation was identified in all patients, of which six were homozygous, two were heterozygous, and one was compound heterozygous with this mutation and a novel heterozygous missense mutation c.488G>A (p.R163H). The levels of PGE2 in urine were much higher than normal in all patients, along with lower PGE-M levels. In conclusion, nine PHO patients were characterized by typical clinical manifestations including digital clubbing, periostosis, and pachydermia. A common mutation and a novel mutation in HPGD gene were identified to be responsible for the disease, and c.310_311delCT mutation is likely to be a hot-spot mutation site for Asian PHO patients.

Supermacroporous polymer monolith prepared with polymeric porogens via viscoelastic phase separation for capillary electrochromatography.

Supermacroporous poly(methacrylic acid-butyl methacrylate-ethylene glycol dimethacrylate) monoliths with the pore size up to 5-10 µm were successfully prepared in a ternary polymeric porogens utilizing viscoelastic effect. High concentration (over 20 mg/mL) of polystyrene (PS) in porogen was used to achieve the desirable characteristics of the monolithic capillary. Modification of the co-porogen composition, i.e., the content of dimethyl sulfoxide and isooctane, enabled tailoring of the supermacropore structure with a wide range of pore size. The effects of the amount of polymer porogen and molecular weights of PS on the formation of supermacropore were also studied. In preliminary applications, the separations of alkyl phenones and alkylbenzenes were achieved on the supermacropore columns using a mode of capillary electrochromatography. The study demonstrated successfully the ability of polymer porogen to form supermacropore monolith via viscoelastic phase separation.

Rapid preparation of molecularly imprinted polymer by frontal polymerization.

Frontal polymerization was successfully applied, for the first time, to obtain molecularly imprinted polymers (MIPs). The method provides a solvent-free polymerization mode, and the reaction can be completed in 30 min. By this approach, MIPs were synthesized using a mixture of levofloxacin (template), methacrylic acid, and divinylbenzene. The effect of template concentration and the amount of comonomer on the imprinting effect of the resulting MIPs was investigated. The textural and morphological parameters of the MIP particles were also characterized by mercury intrusion porosimetry, nitrogen adsorption isotherms, and scanning electron microscopy, providing evidence concerning median pore diameter, pore volumes, and pore size distributions. The levofloxacin-imprinted polymer formed in frontal polymerization mode showed high selectivity, with an imprinting factor of 5.78. The results suggest that frontal polymerization provides an alternative means to prepare MIPs that are difficult to synthesize and may open up new perspectives in the field of MIPs.

Preparation and characterization of grafted imprinted monolith for capillary electrochromatography.

In this paper, a molecularly imprinted polymer (MIP) coating grafted to a trimethylolpropane trimethacrylate (TRIM) core material for CEC was reported. The core monolith was prepared with a solution of 20% (w/w) TRIM in a mixture of porogen and a polymerization precursor, which can generate a stable electroosmotic flow due to the formation of ionizable groups after postpolymerization hydrolization. Graft polymerization took place on the resultant TRIM monolith with a mixture of template, methacrylic acid, and ethylene glycol dimethacrylate. Strong recognition ability (selectivity factor was 5.83) for S-amlodipine and resolution of enatiomers separation (up to 7.99) were obtained on the resulting grafted imprinted monolith in CEC mode. The influence of CEC conditions on chiral separation, including the composition of mobile phase, pH value, and the operating voltages was studied. These results suggest that the method of grafted polymerization reported here allows a rapid development of MIP monolith once core materials with desired properties are available, and is a good alternative to prepare CEC-based monolithic MIPs.